Boron-containing small molecules

ABSTRACT

This invention relates to compounds useful for treating fungal infections, more specifically topical treatment of onychomycosis and/or cutaneous fungal infections. This invention is directed to compounds that are active against fungi and have properties that allow the compound, when placed in contact with a patient, to reach the particular part of the skin, nail, hair, claw or hoof infected by the fungus. In particular the present compounds have physiochemical properties that facilitate penetration of the nail plate.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application a Continuation of U.S. patent application Ser.No. 13/356,488, filed Jan. 23, 2012, which is a continuation of U.S.patent application Ser. No. 12/629,753, filed Dec. 2, 2009, which is adivisional of U.S. patent application Ser. No. 11/505,591 filed Aug. 16,2006, now U.S. Pat. No. 7,767,657, which claims the benefit of U.S.Provisional Patent Application 60/755,227 filed Dec. 30, 2005, and thebenefit of U.S. Provisional Patent Application 60/746,361 filed May 3,2006, all of which are incorporated by reference in their entirety forall purposes. U.S. patent application is also a continuation-in-part ofU.S. patent application Ser. No. 11/357,687 filed Feb. 16, 2006, nowU.S. Pat. No. 7,582,621, which claims the benefit of U.S. ProvisionalPatent Application 60/654,060 filed Feb. 16, 2005, all of which areincorporated by reference in their entirety for all purposes.

BACKGROUND FOR THE INVENTION

Infections of the nail and hoof, known as ungual and/or periungualinfections, pose serious problems in dermatology. These ungual and/orperiungual can be caused by sources such as fungi, viruses, yeast,bacteria and parasites. Onychomycosis is an example of these seriousungual and/or periungual infections and is caused by at least onefungus. Current treatment for ungual and/or periungual infectionsgenerally falls into three categories: systemic administration ofmedicine; surgical removal of all or part of the nail or hoof followedby topical treatment of the exposed tissue; or topical application ofconventional creams, lotions, gels or solutions, frequently includingthe use of bandages to keep these dosage forms in place on the nail orhoof. All of these approaches have major drawbacks. The followingdiscussion is particularly directed to drawbacks associated with currenttreatment of ungual and/or periungual antifungal infections.

Long term systemic (oral) administration of an antifungal agent for thetreatment of onychomycosis is often required to produce a therapeuticeffect in the nail bed. For example, oral treatment with the antifungalcompound terbinafine typically requires administration of 200 to 400mg/day for 12 weeks before any significant therapeutic benefit isrealized. Such long term, high dose systemic therapy can havesignificant adverse effects. For example, terbinafine has been reportedto have liver toxicity effects and reduces testosterone levels in blooddue to adverse effects on the testes. Patient compliance is a problemwith such long term therapies especially those which involve seriousadverse effects. Moreover, this type of long term oral therapy isinconvenient in the treatment of a horse or other ruminants afflictedwith fungal infections of the hoof. Accordingly, the risks associatedwith parenteral treatments generate significant disincentive againsttheir use and considerable patient non-compliance.

Surgical removal of all or part of the nail followed by topicaltreatment also has severe drawbacks. The pain and discomfort associatedwith the surgery and the undesirable cosmetic appearance of the nail ornail bed represent significant problems, particularly for patients moresensitive to physical appearance. Generally, this type of treatment isnot realistic for ruminants such as horses.

Topical therapy has significant problems too. Topical dosage forms suchas creams, lotions, gels etc., can not keep the drug in intimate contactwith the infected area for therapeutically effective periods of time.Bandages have been used to hold drug reservoirs in place in an attemptto enhance absorption of the pharmaceutical agent. However the bandagesare thick, awkward, troublesome and generally lead to poor patientcompliance.

Hydrophilic and hydrophobic film forming topical antifungal solutionshave also been developed. These dosage forms provide improved contactbetween the drug and the nail. Topical formulations for fungal infectiontreatment have largely tried to deliver the drug to the target site (aninfected nail bed) by diffusion across or through the nail.

Nail is more like hair than stratum corneum with respect to chemicalcomposition and permeability. Nitrogen is the major component of thenail attesting to the nail's proteinaceous nature. The total lipidcontent of mature nail is 0.1-1.0%, while the stratum corneum lipid isabout 10% w/w. The nail is 100-200 times thicker than the stratumcorneum and has a very high affinity and capacity for binding andretaining antifungal drugs. Consequently little if any drug penetratesthrough the nail to reach the target site. Because of these reasonstopical therapy for fungal infections have generally been ineffective.

Compounds known as penetration or permeation enhancers are well known inthe art to produce an increase in the permeability of skin or other bodymembranes to a pharmacologically active agent. The increasedpermeability allows an increase in the rate at which the drug permeatesthrough the skin and enters the blood stream. Penetration enhancers havebeen successful in overcoming the impermeability of pharmaceuticalagents through the skin. However, the thin stratum corneum layer of theskin, which is about 10 to 15 cells thick and is formed naturally bycells migrating toward the skin surface from the basal layer, has beeneasier to penetrate than nails. Moreover, known penetration enhancershave not proven to be useful in facilitating drug migration through thenail tissue.

Antimicrobial compositions for controlling bacterial and fungalinfections comprising a metal chelate of 8-hydroxyquinoline and an alkylbenzene sulfonic acid have been shown to be efficacious due to theincreased ability of the oleophilic group to penetrate the lipoid layersof micro-cells. The compounds however, do not effectively increase theability to carry the pharmaceutically active antifungal through thecornified layer or stratum corneum of the skin. U.S. Pat. No. 4,602,011,West et al., Jul. 22, 1986; U.S. Pat. No. 4,766,113, West et al., Aug.23, 1988.

Therefore, there is a need in the art for compounds which caneffectively penetrate the nail. There is also need in the art forcompounds which can effectively treat ungual and/or periungualinfections. These and other needs are addressed by the currentinvention.

Aminoacyl-tRNA synthetases (ARS) are a family of essential enzymes thatattach amino acids to the 3′ terminal adenosine end of tRNAs, thecharged tRNAs are then used by the translation machinery to synthesisproteins from mRNA. Although there are few exceptions, for example inGram-positive bacteria and archaea, most organisms have at least one ARSfor each amino acid. In the case of eukaryotes, they have two ARS, oneis localized to the cytoplasm while the other ARS is located in theorganelle(s). The ARS catalyzes two reactions, as outlined below, thefirst reaction adenylates the amino acid with ATP followed by itstransfer to the 2′- or 3′-hydroxyl of the terminal adenosine of tRNA.

Amino acid (AA)+ATP→AA-AMP+PPi;

AA-AMP+tRNA→tRNA-AA+AMP

The family of 20 ARS fall into two distinct structural classes asdetermined by their crystal structure. Class I, which have a Rossmanlike fold, include the ARS for the following amino acids-arginine,cysteine, glutamate, glutamine, isolelucine, leucine, lysine (in archaeaand some bacteria), valine, methionine, tryptophan and tyrosine. ClassII ARS include the enzymes for the amino acids, alanine, asparagine,aspartate, glycine, histidine, lysine, phenylalanine, proline, serineand threonine. The ARS mediated reaction is the major checkpoint ofspecificity that ensures the correct amino acid is charged to itscognate tRNA. Since some amino acids only differ by a single methylenegroup, for example valine and isoleucine, it has been postulated thatthe specificity of the synthetic reaction alone can't explain theobserved in vivo accuracy of tRNA charging. The synthetic active siteshould be able to exclude amino acids that are not close analogs of thecognate amino acid, but analogous amino acids pose a bigger problem.Therefore to increase specificity, proof-reading and editing must occur.So far nine ARS have been shown to have an editing mechanism thatsignificantly reduces the frequency of mischarged tRNAs. The enzymes forthe following amino acids have been shown to have editingactivity-alanine, isoleucine, leucine, methionine, lysine,phenylalanine, proline, threonine and valine. These ARS can hydrolysethe incorrectly adenylated amino acid AA-AMP (pre-transfer editing) orthe incorrectly charged tRNA (post-transfer editing). To date theisoleucyl, leucyl and valyl-tRNA synthetases have the best-characterizedediting mechanisms; an additional structural domain called theconnective polypeptide I (CP1) inserted in the synthetic domain has beenshown to contain the editing active site. This is located more than 25 Åaway from the synthetic active site, which suggests that both theadenylated amino acid intermediate and amino acid tethered to the 3′ endof the tRNA must be moved from the active site in the synthetic domainto the editing site for the reaction to be proof-read. It has beenpostulated that the 3′ end of the charged tRNA is translocated in asimilar manner to that of the proof-reading mechanism of DNApolymerases. Much less is known about the translocation of theadenylated amino acid. A similar CP1 domain is also present in themethionine and cysteine ARS enzymes, but it is much smaller than thatfound in the valine, isoleucine and leucine enzymes. Despite the absenceof a direct homolog to the CP1-like domain in class II ARS, separateediting domains have been found in the enzymes for proline andthreonine. Although editing is important to ensure the correct chargingof tRNAs, it is not essential for viability and is not required for thesynthesis of charged tRNAs. For example, in Escherichia coli, in which10 amino acids in the editing domain of isoleucyl-tRNA synthetase werechanged to alanine, the resulting mutant was still viable, although itdid have many pleiotropic effects, including a noticeable cell growthdefect.

In spite of significant homologies between human, bacterial and fungalARS there are a number of compounds that have been developed asanti-infectives. The most notable example of an ARS inhibitor is thecommercial antibiotic mupirocin (pseudomonic acid), which is sold underthe label Bactroban. Mupirocin specifically inhibits bacterialisoleucyl-tRNA synthetases, while its activity against the human homologis more than 1,000 times less active. Mupirocin binds specifically tothe synthetic active site and mutants that are resistant to this drughave mutations in the synthetic domain of leucyl-tRNA synthetase.Likewise, reveromycin A inhibits the eukaryotic isoleucyl-tRNAsynthetases: Saccharomyces cerevisiae resistance mutants have mutationsin the synthetic domain. So far all attempts to develop better ARSinhibitors than mupirocin, an isoleucine-adenylate analogue, have reliedon inhibiting the synthetic reactions.

Since it has been previously thought not to be essential for thesynthesis of charged tRNAs, the editing domain of tRNA synthetases hasnot been thought a promising target for drug development. Data frommutational analysis of the ARS editing domains tend to suggest thatinhibition of the editing mechanism leads only to an increase inmischarged tRNAs and does not lead to cell death. Compounds that areactive against, and specific for, the editing domain of the tRNAsynthetase would provide access to a new class of antimicrobialtherapeutics to augment the arsenal of agents currently in use. Quitesurprisingly, the present invention provides such compounds and methodsof using these compounds.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a structure according to thefollowing formula:

in which R¹ and R² are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. R¹ and R², together with theatoms to which they are attached, can be optionally joined to form a 4-to 7-membered ring. Z1 is a member selected from

R^(3a) and R^(4a) are members independently selected from H, cyano,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. R⁵ is a member selected fromhalogen and OR⁸. R⁸ is a member selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. A is a member selected from CR^(9a) and N. Dis a member selected from CR^(10a) and N. E is a member selected fromCR^(11a) and N. G is a member selected from CR^(12a) and N. R^(9a),R^(10a), R^(11a) and R^(12a) are members independently selected from H,OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*, —S(O)₂NR*R**, nitro, halogen,cyano, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. Each R* and R** are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl.R^(9a) and R^(10a), along with the atoms to which they are attached, areoptionally joined to form a ring. R^(10a) and R^(11a), along with theatoms to which they are attached, are optionally joined to form a ring.R^(11a) and R^(12a), along with the atoms to which they are attached,are optionally joined to form a ring. The combination of nitrogens(A+D+E+G) is an integer selected from 0 to 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of minimum inhibitory concentration (MIC) data ofcyclic boronic esters against various fungi.

FIG. 2A displays minimum inhibitory concentration (MIC) for C10,ciclopirox, terbinafine, fluconazole and itraconazole (comparator drugs)against 19 test strains of fungi.

FIG. 2B displays minimum fungicidal concentration (MFC) for C10,ciclopirox, terbinafine and itraconazole (comparator drugs) against 2test strains of fungi.

FIG. 3 displays a comparison of Normalized C10 and Ciclopirox Equivalentin Each Part of Nail Plate Samples after 14-day Treatment.

FIG. 4 displays a comparison of C10 and Ciclopirox Equivalent in CottonBall Supporting Bed Samples after 14-day Treatment.

FIG. 5 displays the results of a placebo for C10 (50:50 propylene glycoland ethyl acetate) applied per day over five days. Full carpet growth ofthe organism T. rubrum was observed.

FIG. 6 displays the results of a 40 μL/cm² aliquot of C10 10% w/vsolution applied per day over five days. Zones of inhibition (in theorder of the cells shown in the figure) of 100%, 67%, 46%, 57%, 38% and71% were observed for the growth of T. rubrum. Green arrow indicates themeasurement of zone of inhibition.

FIG. 7 displays the results of a 40 μL/cm² aliquot of C10 10% w/vsolution applied per day over five days. Zones of inhibition (in theorder of the cells shown in the figure) of 74%, 86%, 100%, 82%, 100% and84% were observed for the growth of T. rubrum.

FIG. 8 displays the results of a 40 μL/cm² aliquot of 8% ciclopirox inw/w commercial lacquer applied per day over five days. No zone ofinhibition observed; full carpet growth of T. rubrum.

FIG. 9 displays the results of a 40 μL/cm² aliquot of 5% amorolfine w/vin commercial lacquer applied per day over five days. No zone ofinhibition observed; full carpet growth of T. rubrum.

FIG. 10 Amino acid sequences for leucyl-tRNA synthetase editing domainsand nucleotide sequences for tRNA-Leu and tRNA-Ile. (A) Amino acidsequences for leucyl-tRNA synthetase editing domain from S. cerivisiaein wild type (SEQ ID NO: 1) and over-expressing form (SEQ ID NO: 2); (B)Amino acid sequences for leucyl-tRNA synthetase editing domains fromindicated species; (C) Genomic nucleotide sequence for tRNA-leu andtRNA-ile from S. cerivisiae; in one embodiment of the invention, anaminoacyl tRNA synthetase will bind to the transcribed and methylatedproducts for which these sequences serve as a template; (D) tRNA-Leunucleotide sequences from indicated species.

FIG. 11 displays structures of cyclic boronic esters.

FIG. 12 displays different structures for portions of the compounds ofthe invention.

FIG. 13 Effect of ATP on binding of C10 to cdc60. The binding assay wasconducted with an initial [C10] concentration of approximately 72-79 μM(pre-equilibrium).

FIG. 14 Binding curve of cdc60 against concentration of free [C10].

FIG. 15 Data from PPi exchange reaction experiment to determine rate ofediting in the presence and absence of C10.

FIG. 16 Data from an aminoacylation experiment showing the effect of C10at different concentrations on the aminoacylation of tRNA^(leu).

FIG. 17 Results from post transfer editing assay conducted in S.cerevisiae at differing concentrations of C10 across a range of timepoints.

FIG. 18 displays the names of exemplary compounds of the invention.

FIG. 19 displays exemplary compounds of the invention.

FIG. 20 displays exemplary compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Abbreviations

The abbreviations used herein generally have their conventional meaningwithin the chemical and biological arts.

“Compound of the invention,” as used herein refers to the compoundsdiscussed herein, pharmaceutically acceptable salts and prodrugs ofthese compounds.

“Boron containing compounds”, as used herein, refers to the compounds ofthe invention that contain boron as part of their chemical formula.

MIC, or minimum inhibitory concentration, is the point where thecompound stops more than 50% of cell growth, preferably 60% of cellgrowth, preferably 70% of cell growth, preferably 80% of cell growth,preferably 90% of cell growth, relative to an untreated control.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “poly” as used herein means at least 2. For example, apolyvalent metal ion is a metal ion having a valency of at least 2.

“Moiety” refers to the radical of a molecule that is attached to anothermoiety.

The symbol

, whether utilized as a bond or displayed perpendicular to a bond,indicates the point at which the displayed moiety is attached to theremainder of the molecule.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom. In anexemplary embodiment, the heteroatoms can be selected from the groupconsisting of B, O, N and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. The heteroatom(s) B, O, N and S may be placed at anyinterior position of the heteroalkyl group or at the position at whichthe alkyl group is attached to the remainder of the molecule. Examplesinclude, but are not limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃. Similarly, the term “heteroalkylene” by itself or as partof another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —RC(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms. In an exemplary embodiment, theheteroatom is selected from B, N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, 6-quinolyl, dioxaborolane, dioxaborinane and dioxaborepane.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂ in a numberranging from zero to (2m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″, R′″ and R′″ each preferably independentlyrefer to hydrogen, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted aryl, e.g., aryl substituted with 1-3 halogens,substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, orarylalkyl groups. When a compound of the invention includes more thanone R group, for example, each of the R groups is independently selectedas are each R′, R″, R″ and R″″ groups when more than one of these groupsis present. When R′ and R″ are attached to the same nitrogen atom, theycan be combined with the nitrogen atom to form a 5-, 6-, or 7-memberedring. For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃,—CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R′, R″, R′″ and R″″ are preferably independentlyselected from hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′-, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′-, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′-.The substituents R′, R′, R″ and R′″ are preferably independentlyselected from hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

“Ring” as used herein, means a substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. A ringincludes fused ring moieties. The number of atoms in a ring is typicallydefined by the number of members in the ring. For example, a “5- to7-membered ring” means there are 5 to 7 atoms in the encirclingarrangement. The ring optionally included a heteroatom. Thus, the term“5- to 7-membered ring” includes, for example pyridinyl and piperidinyl.The term “ring” further includes a ring system comprising more than one“ring”, wherein each “ring” is independently defined as above.

As used herein, the term “heteroatom” includes atoms other than carbon(C) and hydrogen (H). Examples include oxygen (O), nitrogen (N) sulfur(S), silicon (Si), germanium (Ge), aluminum (Al) and boron (B).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted cycloalkyl and substituted or unsubstitutedheterocycloalkyl groups.

The term “derived from” includes its plain language meaning and alsorefers to a molecule that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%,65%, or 60% homologous to a referenced molecule. The molecules referredto in this definition include chains of RNA or DNA, oligonucleotides,polypeptides, or proteins of any length and composition.

The term “immunological marker” includes oligonucleotides, proteins,antibodies, peptides, polypeptides, enzymes, or any other molecule ableto induce an immune response in appropriate animals or cells or to bindwith specific antibodies.

The term “noncognate” is meant to encompass both the singular and pluralforms of the word, i.e. the phrase “noncognate amino acid” comprises oneor more amino acids.

By “effective” amount of a drug, formulation, or permeant is meant asufficient amount of a active agent to provide the desired local orsystemic effect. A “Topically effective,” “Cosmetically effective,”“pharmaceutically effective,” or “therapeutically effective” amountrefers to the amount of drug needed to effect the desired therapeuticresult.

“Topically effective” refers to a material that, when applied to theskin, nail, hair, claw or hoof produces a desired pharmacological resulteither locally at the place of application or systemically as a resultof transdermal passage of an active ingredient in the material.

“Cosmetically effective” refers to a material that, when applied to theskin, nail, hair, claw or hoof, produces a desired cosmetic resultlocally at the place of application of an active ingredient in thematerial.

The term “pharmaceutically acceptable salts” is meant to include saltsof the compounds of the invention which are prepared with relativelynontoxic acids or bases, depending on the particular substituents foundon the compounds described herein. When compounds of the presentinvention contain relatively acidic functionalities, base addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science 66: 1-19 (1977)). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompounds in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds or complexesdescribed herein readily undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” refers to any formulation or carrier medium thatprovides the appropriate delivery of an effective amount of a activeagent as defined herein, does not interfere with the effectiveness ofthe biological activity of the active agent, and that is sufficientlynon-toxic to the host or patient. Representative carriers include water,oils, both vegetable and mineral, cream bases, lotion bases, ointmentbases and the like. These bases include suspending agents, thickeners,penetration enhancers, and the like. Their formulation is well known tothose in the art of cosmetics and topical pharmaceuticals. Additionalinformation concerning carriers can be found in Remington: The Scienceand Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins(2005) which is incorporated herein by reference.

“Pharmaceutically acceptable topical carrier” and equivalent terms referto pharmaceutically acceptable carriers, as described herein above,suitable for topical application. An inactive liquid or cream vehiclecapable of suspending or dissolving the active agent(s), and having theproperties of being nontoxic and non-inflammatory when applied to theskin, nail, hair, claw or hoof is an example of apharmaceutically-acceptable topical carrier. This term is specificallyintended to encompass carrier materials approved for use in topicalcosmetics as well.

The term “pharmaceutically acceptable additive” refers to preservatives,antioxidants, fragrances, emulsifiers, dyes and excipients known or usedin the field of drug formulation and that do not unduly interfere withthe effectiveness of the biological activity of the active agent, andthat is sufficiently non-toxic to the host or patient. Additives fortopical formulations are well-known in the art, and may be added to thetopical composition, as long as they are pharmaceutically acceptable andnot deleterious to the epithelial cells or their function. Further, theyshould not cause deterioration in the stability of the composition. Forexample, inert fillers, anti-irritants, tackifiers, excipients,fragrances, opacifiers, antioxidants, gelling agents, stabilizers,surfactant, emollients, coloring agents, preservatives, bufferingagents, other permeation enhancers, and other conventional components oftopical or transdermal delivery formulations as are known in the art.

The terms “enhancement,” “penetration enhancement” or “permeationenhancement” relate to an increase in the permeability of the skin,nail, hair, claw or hoof to a drug, so as to increase the rate at whichthe drug permeates through the skin, nail, hair, claw or hoof. Theenhanced permeation effected through the use of such enhancers can beobserved, for example, by measuring the rate of diffusion of the drugthrough animal or human skin, nail, hair, claw or hoof using a diffusioncell apparatus. A diffusion cell is described by Merritt et al.Diffusion Apparatus for Skin Penetration, J of Controlled Release, 1(1984) pp. 161-162. The term “permeation enhancer” or “penetrationenhancer” intends an agent or a mixture of agents, which, alone or incombination, act to increase the permeability of the skin, nail, hair orhoof to a drug.

The term “excipients” is conventionally known to mean carriers, diluentsand/or vehicles used in formulating drug compositions effective for thedesired use.

The term “topical administration” refers to the application of apharmaceutical agent to the external surface of the skin, nail, hair,claw or hoof, such that the agent crosses the external surface of theskin, nail, hair, claw or hoof and enters the underlying tissues.Topical administration includes application of the composition to intactskin, nail, hair, claw or hoof, or to an broken, raw or open wound ofskin, nail, hair, claw or hoof. Topical administration of apharmaceutical agent can result in a limited distribution of the agentto the skin and surrounding tissues or, when the agent is removed fromthe treatment area by the bloodstream, can result in systemicdistribution of the agent.

The term “transdermal delivery” refers to the diffusion of an agentacross the barrier of the skin, nail, hair, claw or hoof resulting fromtopical administration or other application of a composition. Thestratum corneum acts as a barrier and few pharmaceutical agents are ableto penetrate intact skin. In contrast, the epidermis and dermis arepermeable to many solutes and absorption of drugs therefore occurs morereadily through skin, nail, hair, claw or hoof that is abraded orotherwise stripped of the stratum corneum to expose the epidermis.Transdermal delivery includes injection or other delivery through anyportion of the skin, nail, hair, claw or hoof or mucous membrane andabsorption or permeation through the remaining portion. Absorptionthrough intact skin, nail, hair, claw or hoof can be enhanced by placingthe active agent in an appropriate pharmaceutically acceptable vehiclebefore application to the skin, nail, hair, claw or hoof. Passivetopical administration may consist of applying the active agent directlyto the treatment site in combination with emollients or penetrationenhancers. As used herein, transdermal delivery is intended to includedelivery by permeation through or past the integument, i.e. skin, nail,hair, claw or hoof.

The term “microbial infection” refers to any infection of a host tissueby an infectious agent including, but not limited to, viruses, bacteria,mycobacteria, fungus and parasites (see, e.g., Harrison's Principles ofInternal Medicine, pp. 93-98 (Wilson et al., eds., 12th ed. 1991);Williams et al., J. of Medicinal Chem. 42:1481-1485 (1999), herein eachincorporated by reference in their entirety).

“Biological medium,” as used herein refers to both in vitro and in vivobiological milieus. Exemplary in vitro “biological media” include, butare not limited to, cell culture, tissue culture, homogenates, plasmaand blood. In vivo applications are generally performed in mammals,preferably humans.

“Inhibiting” and “blocking,” are used interchangeably herein to refer tothe partial or full blockade of an editing domain of a tRNA synthetase.

“Ventral/intermediate center” as used herein refers to powdered nailsamples drilled from the center of the inner surface (facing the nailbed) approximately 0.3-0.5 mm in depth to the surface. The area isbeneath the dosed site of the nail place but does not include dosedsurface (dorsal nail surface).

“Ventral/intermediate center” as used herein refers to the immediatearea of dosed site.

“Remainder nail” as used herein refers to the remaining part of the nailthat has not been dosed.

“Supporting bed” as used herein refers to the cotton ball placed withinthe Teflon chamber of the diffusion cell to provide moisture to the nailplate and also to receive chemicals penetrating through the nail plate.

“Surfacing washing” as used herein refers to ethanol (or other organicsolvents) and soap/water washing on the surface of the dosed site.

“Cell washing” as used herein, refers to ethanol (or other organicsolvents) and soap/water wash of the inside of the diffusion cell.

A “human nail unit”, as defined herein, can be the nail plate, the nailbed, proximal nail fold, lateral nail fold and combinations thereof.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include triflate, chloro, bromoand iodo groups; sulfonic ester groups, such as mesylate, tosylate,brosylate, nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl, trichloroacetyl ortrifluoroacetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and thelike.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

Boron is able to form dative bonds with oxygen, sulfur or nitrogen undersome circumstances in this invention. Dative bonds are usually weakerthan covalent bonds. In situations where a boron is covalently bonded toat least one oxygen, sulfur or nitrogen, and is at the same timedatively bonded to an oxygen, sulfur or nitrogen, respectively, thedative bond and covalent bond between the boron and the two identicalheteroatoms can interconvert or be in the form of a resonance hybrid.There is potential uncertainty surrounding the exact nature and extentof electron sharing in these situations. The structures supplied are notintended to include any and all possible bonding scenarios between boronand the atom to which it is bound. Non limiting examples of these bondsare as follows:

“Salt counterion”, as used herein, refers to positively charged ionsthat associate with a compound of the invention when the boron is fullynegatively or partially negatively charged. Examples of salt counterionsinclude H⁺, H₃O⁺, ammonium, potassium, calcium, magnesium and sodium.

The compounds comprising a boron bonded to a carbon and threeheteroatoms (such as three oxygens described in this section) canoptionally contain a fully negatively charged boron or partiallynegatively charged boron, due to the nature of the dative bond betweenthe boron and one of the oxygens. Due to the negative charge, apositively charged counterion may associate with this compound, thusforming a salt. Examples of positively charged counterions include H⁺,H₃O⁺, calcium, sodium, ammonium, potassium. The salts of these compoundsare implicitly contained in descriptions of these compounds.

The present invention also encompasses compounds that are poly- ormulti-valent species, including, for example, species such as dimers,trimers, tetramers and higher homologs of the compounds of use in theinvention or reactive analogues thereof. For example, dimers of C10 canform under the following conditions:

In another example, dimers of C17 can form under the followingconditions:

The present invention also encompasses compounds that are anhydrides ofthe cyclic boronic esters are synthesized by subjecting these compoundsto dehydrating conditions. Examples of these anhydrides are providedbelow:

Trimers of the compounds of the invention are also produced. Forexample, trimers of acyclic boronic esters can be formed as follows:

Polymers of the compounds of the invention are also produced through theremoval of certain protecting groups in strong acid. For example,trimers of acyclic boronic esters can be formed as follows:

Also of use in the present invention are compounds that are poly- ormulti-valent species, including, for example, species such as dimers,trimers, tetramers and higher homologs of the compounds of use in theinvention or reactive analogues thereof. The poly- and multi-valentspecies can be assembled from a single species or more than one speciesof the invention. For example, a dimeric construct can be “homo-dimeric”or “heterodimeric.” Moreover, poly- and multi-valent constructs in whicha compound of the invention or a reactive analogue thereof, is attachedto an oligomeric or polymeric framework (e.g., polylysine, dextran,hydroxyethyl starch and the like) are within the scope of the presentinvention. The framework is preferably polyfunctional (i.e. having anarray of reactive sites for attaching compounds of use in theinvention). Moreover, the framework can be derivatized with a singlespecies of the invention or more than one species of the invention.

Moreover, the present invention includes the use of compounds within themotif set forth in the formulae contained herein, which arefunctionalized to afford compounds having water-solubility that isenhanced relative to analogous compounds that are not similarlyfunctionalized. Thus, any of the substituents set forth herein can bereplaced with analogous radicals that have enhanced water solubility.For example, it is within the scope of the invention to replace ahydroxyl group with a diol, or an amine with a quaternary amine, hydroxyamine or similar more water-soluble moiety. In a preferred embodiment,additional water solubility is imparted by substitution at a site notessential for the activity towards the editing domain of the compoundsset forth herein with a moiety that enhances the water solubility of theparent compounds. Methods of enhancing the water-solubility of organiccompounds are known in the art. Such methods include, but are notlimited to, functionalizing an organic nucleus with a permanentlycharged moiety, e.g., quaternary ammonium, or a group that is charged ata physiologically relevant pH, e.g. carboxylic acid, amine. Othermethods include, appending to the organic nucleus hydroxyl- oramine-containing groups, e.g. alcohols, polyols, polyethers, and thelike. Representative examples include, but are not limited to,polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art. See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991.

II. Introduction

The present invention provides novel boron compounds and methods for thepreparation of these molecules. The invention further provides boroncompounds as analogs comprising a functional moiety, such as a drugmoiety and methods of use for said analogs.

III. The Compounds III.a) Cyclic Boronic Esters

In a first aspect, the invention provides a compound having a structureaccording to Formula I:

wherein B is boron. R^(1a) is a member selected from a negative charge,a salt counterion, H, cyano, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl. M isa member selected from oxygen, sulfur and NR^(2a). R^(2a) is a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. J is amember selected from (CR^(3a)R^(4a))_(n1) and CR^(5a). R^(3a), R^(4a),and R^(5a) are members independently selected from H, cyano, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. The index n1 is an integer selected from 0 to2. W is a member selected from C═O (carbonyl), (CR^(6a)R^(7a))_(m1) andCR^(5a). R^(6a), R^(7a), and R^(8a) are members independently selectedfrom H, cyano, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. Theindex m1 is an integer selected from 0 and 1. A is a member selectedfrom CR^(9a) and N. D is a member selected from CR^(10a) and N. E is amember selected from CR^(11a) and N. G is a member selected fromCR^(12a) and N. R^(9a), R^(10a), R^(11a) and R^(12a) are membersindependently selected from H, OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*,—S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, nitro, halogen, cyano,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. Each R* and R** are membersindependently selected from H, nitro, halogen, cyano, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. The combination of nitrogens (A+D+E+G) is aninteger selected from 0 to 3. A member selected from R^(3a), R^(4a) andR^(5a) and a member selected from R^(6a), R^(7a) and R^(8a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring. R^(3a) and R^(4a), together with the atoms towhich they are attached, are optionally joined to form a 4 to 7 memberedring. R^(6a) and R^(7a), together with the atoms to which they areattached, are optionally joined to form a 4 to 7 membered ring. R^(9a)and R^(10a), together with the atoms to which they are attached, areoptionally joined to form a 4 to 7 membered ring. R^(10a) and R^(11a)together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring. R^(11a) and R^(12a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring.

In an exemplary embodiment, the compound has a structure according toFormula (Ia):

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberindependently selected from H, cyano, substituted or unsubstitutedmethyl, substituted or unsubstituted ethyl, trifluoromethyl, substitutedor unsubstituted hydroxymethyl, substituted or unsubstitutedhydroxyalkyl, substituted or unsubstituted benzyl, substituted orunsubstituted phenyl, substituted or unsubstituted mercaptomethyl,substituted or unsubstituted mercaptoalkyl, substituted or unsubstitutedaminomethyl, substituted or unsubstituted alkylaminomethyl, substitutedor unsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted indolyl and substituted orunsubstituted amido. In another exemplary embodiment, each R^(3a) andR^(4a) is a member independently selected from cyano, substituted orunsubstituted methyl, substituted or unsubstituted ethyl,trifluoromethyl, substituted or unsubstituted hydroxymethyl, substitutedor unsubstituted hydroxyalkyl, substituted or unsubstituted benzyl,substituted or unsubstituted phenyl, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted indolyl, substituted or unsubstituted amido.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. In another exemplary embodiment,R^(3a) and R^(4a) is a member selected from methyl, ethyl, propyl,isopropyl, butyl, t-butyl, phenyl and benzyl. In another exemplaryembodiment, R^(3a) is H and R^(4a) is a member selected from methyl,ethyl, propyl, isopropyl, butyl, t-butyl, phenyl and benzyl. In anotherexemplary embodiment, R^(3a) is H and R^(4a)H.

In another exemplary embodiment, each R^(9a), R^(10a), R^(11a) andR^(12a) is a member independently selected from H, OR*, NR*R**, SR*,—S(O)R*, —S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**,halogen, cyano, nitro, substituted or unsubstituted methoxy, substitutedor unsubstituted methyl, substituted or unsubstituted ethoxy,substituted or unsubstituted ethyl, trifluoromethyl, substituted orunsubstituted hydroxymethyl, substituted or unsubstituted hydroxyalkyl,substituted or unsubstituted benzyl, substituted or unsubstitutedphenyl, substituted or unsubstituted phenyloxy, substituted orunsubstituted phenyl methoxy, substituted or unsubstitutedthiophenyloxy, substituted or unsubstituted pyridinyloxy, substituted orunsubstituted pyrimidinyloxy, substituted or unsubstituted benzylfuran,substituted or unsubstituted methylthio, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted phenylthio, substituted or unsubstitutedthiophenylthio, substituted or unsubstituted phenyl methylthio,substituted or unsubstituted pyridinylthio, substituted or unsubstitutedpyrimidinylthio, substituted or unsubstituted benzylthiofuranyl,substituted or unsubstituted phenylsulfonyl, substituted orunsubstituted benzylsulfonyl, substituted or unsubstitutedphenylmethylsulfonyl, substituted or unsubstituted thiophenylsulfonyl,substituted or unsubstituted pyridinylsulfonyl, substituted orunsubstituted pyrimidinylsulfonyl, substituted or unsubstitutedsulfonamidyl, substituted or unsubstituted phenylsulfinyl, substitutedor unsubstituted benzylsulfinyl, substituted or unsubstitutedphenylmethylsulfinyl, substituted or unsubstituted thiophenylsulfinyl,substituted or unsubstituted pyridinylsulfinyl, substituted orunsubstituted pyrimidinylsulfinyl, substituted or unsubstituted amino,substituted or unsubstituted alkylamino, substituted or unsubstituteddialkylamino, substituted or unsubstituted trifluoromethylamino,substituted or unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted benzylamino, substituted or unsubstituted phenylamino,substituted or unsubstituted thiophenylamino, substituted orunsubstituted pyridinylamino, substituted or unsubstitutedpyrimidinylamino, substituted or unsubstituted indolyl, substituted orunsubstituted morpholino, substituted or unsubstituted alkylamido,substituted or unsubstituted arylamido, substituted or unsubstitutedureido, substituted or unsubstituted carbamoyl, and substituted orunsubstituted piperizinyl. In an exemplary embodiment, R^(9a), R^(10a),R^(11a) and R^(12a) are selected from the previous list of substituentswith the exception of —C(O)R*, —C(O)OR*, —C(O)NR*R**.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy,4-fluorobenzyloxy, unsubstituted phenyl, unsubstituted benzyl. In anexemplary embodiment, R^(9a) is H and R^(12a) is H.

In an exemplary embodiment, the compound according to Formula (I) orFormula (Ia) is a member selected from:

In an exemplary embodiment, the compound has a structure according toone of Formulae I-Io with substituent selections for R^(9a), R^(10a),R^(11a) and R^(12a) including all the possibilities contained inparagraph 106 except for H. In an exemplary embodiment, the compound hasa structure according to one of Formulae Ib-Io with substituentselections for R^(9a), R^(10a), R^(11a) and R^(12a) including all thepossibilities contained in paragraph 107 except for H.

In an exemplary embodiment, the compound has a formula according toFormulae (Ib)-(Ie) wherein R^(1a) is a member selected from H, anegative charge and a salt counterion and the remaining R group (R^(9a)in Ib, R^(10a) in Ic, R^(1a) in Id, and R^(12a) in le) is a memberselected from fluoro, chloro R′, bromo, nitro, cyano, amino, methyl,hydroxylmethyl, trifluoromethyl, methoxy, trifluoromethyoxy, ethyl,diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidinyl,piperizino, piperizinyl, piperizinocarbonyl, piperizinylcarbonyl,carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy, carboxymethoxy,thiophenyl, 3-(butylcarbonyl) phenylmethoxy, 1H-tetrazol-5-yl,1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-, 1-ethoxycarbonyl-,carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-, thiophen-3-yl,thiophen-3-ylthio, 4-fluorophenylthio, butylcarbonylphenylmethoxy,butylcarbonylphenylmethyl, butylcarbonylmethyl,1-(piperidin-1-yl)carbonyl)methyl, 1-(piperidin-1-yl)carbonyl)methoxy,1-(piperidin-2-yl)carbonyl)methoxy, 1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy and4-fluorobenzyloxy.

In an exemplary embodiment, the compound has a formula according toFormulae (If)-(Ik) wherein R^(1a) is a member selected from H, anegative charge and a salt counterion and each of the remaining two Rgroups (R^(9a) and R^(10a) in If, R^(9a) and R^(11a) in Ig, R^(9a) andR^(12a) in Ih, R^(10a) and R^(11a) in Ii, R^(10a) and R^(12a) in Ij,R^(11a) and R^(12a) in Ik) is a member independently selected fromfluoro, chloro, bromo, nitro, cyano, amino, methyl, hydroxylmethyl,trifluoromethyl, methoxy, trifluoromethyoxy, ethyl, diethylcarbamoyl,pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidinyl, piperizino,piperizinyl, piperizinocarbonyl, piperizinylcarbonyl, carboxyl,1-tetrazolyl, 1-ethoxycarbonylmethoxy, carboxymethoxy, thiophenyl,3-(butylcarbonyl)phenylmethoxy, 1H-tetrazol-5-yl,1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-, 1-ethoxycarbonyl-,carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-, thiophen-3-yl,thiophen-3-ylthio, 4-fluorophenylthio, butylcarbonylphenylmethoxy,butylcarbonylphenylmethyl, butylcarbonylmethyl,1-(piperidin-1-yl)carbonyl)methyl, 1-(piperidin-1-yl)carbonyl)methoxy,1-(piperidin-2-yl)carbonyl)methoxy, 1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy.

In an exemplary embodiment, the compound has a formula according toFormulae (Il)-(Io) wherein R^(1a) is a member selected from H, anegative charge and a salt counterion and each of the remaining three Rgroups (R^(9a), R^(10a), R^(11a) in (Il), R^(9a) R^(10a), R^(12a) in(Im), R^(9a), R^(11a), R^(12a) in (In), R¹⁰, R^(11a), R^(12a) in (Io))is a member independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy.

In an exemplary embodiment, there is a proviso that the compound cannotbe a member selected from FIG. 11. In another exemplary embodiment,there is a proviso that the compound cannot be a member selected fromC₁-C₄₀.

In another exemplary embodiment, there is a proviso that the compoundcannot have a structure according to Formula (Ix):

wherein R^(7b) is a member selected from H, methyl, ethyl and phenyl.R^(10b) is a member selected from H, OH, NH₂, SH, halogen, substitutedor unsubstituted phenoxy, substituted or unsubstituted phenylalkyloxy,substituted or unsubstituted phenylthio and substituted or unsubstitutedphenylalkylthio. R^(11b) is a member selected from H, OH, NH₂, SH,methyl, substituted or unsubstituted phenoxy, substituted orunsubstituted phenylalkyloxy, substituted or unsubstituted phenylthioand substituted or unsubstituted phenylalkylthio. In another exemplaryembodiment, there is a proviso that the compound cannot have a structureaccording to Formula (Ix) wherein R^(1b) is a member selected from anegative charge, H and a salt counterion. In another exemplaryembodiment, there is a proviso that the compound cannot have a structureaccording to Formula (Ix) wherein R^(10b) and R^(11b) are H. In anotherexemplary embodiment, there is a proviso that the compound cannot have astructure according to Formula (Ix) wherein one member selected fromR^(10b) and R^(11b) is H and the other member selected from R^(10b) andR^(11b) is a member selected from halo, methyl, cyano, methoxy,hydroxymethyl and p-cyanophenyloxy. In another exemplary embodiment,there is a proviso that the compound cannot have a structure accordingto Formula (Ix) wherein R^(10b) and R^(11b) are members independentlyselected from fluoro, chloro, methyl, cyano, methoxy, hydroxymethyl, andp-cyanophenyl. In another exemplary embodiment, there is a proviso thatthe compound cannot have a structure according to Formula (Ix) whereinR^(1b) is a member selected from a negative charge, H and a saltcounterion; R^(7b) is H; R^(10b) is F and R^(11b) is H. In anotherexemplary embodiment, there is a proviso that the compound cannot have astructure according to Formula (Ix) wherein R^(11b) and R^(12b), alongwith the atoms to which they are attached, are joined to form a phenylgroup. In another exemplary embodiment, there is a proviso that thecompound cannot have a structure according to Formula (Ix) whereinR^(1b) is a member selected from a negative charge, H and a saltcounterion; R^(7b) is H; R^(10b) is 4-cyanophenoxy; and R^(11b) is H.

In another exemplary embodiment, there is a proviso that the compoundcannot have a structure according to Formula (Iy)

wherein R^(10b) is a member selected from H, halogen, CN and substitutedor unsubstituted C₁₋₄ alkyl.

In another exemplary embodiment, there is a proviso that a structuredoes not have the which is a member selected from Formulae (I) to (Io)at least one member selected from R^(3a), R^(4a), R^(5a), R^(6a),R^(7a), R^(8a), R^(9a), R^(10a), R^(11a) and R^(12a) is nitro, cyano orhalogen. In another exemplary embodiment, there is a proviso that when Mis oxygen, W is a member selected from (CR^(3a)R^(4a))_(n1), wherein n1is 0, J is a member selected from (CR^(6a)R^(7a))_(m1), wherein m1 is 1,A is CR^(9a), D is CR^(10a), E is CR^(11a), G is CR^(12a), the R^(9a) isnot halogen, methyl, ethyl, or optionally joined with R^(10a) to form aphenyl ring; R^(10a) is not unsubstituted phenoxy, C(CH₃)₃, halogen,CF₃, methoxy, ethoxy, or optionally joined with R^(9a) to form a phenylring; R^(11a) is not halogen or optionally joined with R^(10a) to form aphenyl ring; and R^(12a) is not halogen. In another exemplaryembodiment, there is a proviso that when M is oxygen, W is a memberselected from (CR^(3a)R^(4a))_(n1), wherein n1 is 0, J is a memberselected from (CR^(6a)R^(7a))_(m), wherein m1 is 1, A is CR^(9a), D isCR^(10a), E is CR^(11a), G1 is CR^(12a), then neither R^(6a) nor R^(7a)are halophenyl. In another exemplary embodiment, there is a proviso thatwhen M is oxygen, W is a member selected from (CR^(3a)R^(4a))_(n1),wherein n1 is 0, J is a member selected from (CR^(6a)R^(7a))_(m),wherein m1 is 1, A is CR^(9a), D is CR^(10a), E is CR^(11a), G isCR^(12a), and R^(9a), R^(10a) and R^(11a) are H, then R^(6a), R^(7a) andR^(12a) are not H. In another exemplary embodiment, there is a provisothat when M is oxygen wherein n1 is 1, J is a member selected from(CR^(6a)R^(7a))_(m), wherein m1 is 0, A is CR^(9a), D is CR^(10a), E isCR^(11a), G is CR^(12a), R^(9a) is H, R^(10a) is H, R^(11a) is H, R^(6a)is H, R^(7a) is H, R^(12a) is H, then W is not C═O (carbonyl). Inanother exemplary embodiment, there is a proviso that when M is oxygen,W is CR^(5a), J is CR^(8a), A is CR^(9a), D is CR^(10a), E is CR^(11a),G is CR^(12a), R^(6a), R^(7a), R^(9a), R^(10a), R^(11a) and R^(12a) areH, then R^(5a) and R^(8a), together with the atoms to which they areattached, do not form a phenyl ring.

In an exemplary embodiment, the compound of the invention has astructure which is a member selected from:

in which q is a number between 0 and 1. R^(g) is halogen. R^(a), R^(b),R^(c), R^(d) and R^(e) are members independently selected from a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. In anexemplary embodiment, there is a proviso that the compound is not amember selected from

In an exemplary embodiment, the compound has a structure is a memberselected from:

In an exemplary embodiment, R^(a), R^(d) and R^(e) are each membersindependently selected from:

In an exemplary embodiment, R^(b) and R^(c) are members independentlyselected from H, methyl,

In another exemplary embodiment, R^(b) is H and R^(o) is a memberselected from H, methyl,

In another exemplary embodiment, R^(b) and R^(c) are, together with thenitrogen to which they are attached, optionally joined to form a memberselected from

In an exemplary embodiment, R^(a) is a member selected from

In an exemplary embodiment, R^(d) is a member selected from

In an exemplary embodiment, R^(e) is a member selected from

In an exemplary embodiment, the compound is a member selected from

In an exemplary embodiment, the compound has a structure which isdescribed in FIG. 19. In an exemplary embodiment, the compound has astructure which is described in FIG. 20.

In an exemplary embodiment, the compound has a structure according to amember selected from Formulae I(b), I(c), I(d), and I(e) wherein saidremaining R group (R^(9a) for I(b), R^(10a) for I(c), R^(11a) for I(d)and R^(12a) for I(e)) is carboxymethoxy.

In an exemplary embodiment, the compound has a structure which is amember selected from Formulae (If)-(Ik), wherein either R^(9a) orR^(10a) for Formula (If), either R^(9a) or R^(11a) for Formula (Ig),either R^(9a) or R^(12a) for Formula (Ih), either R^(10a) or R^(11a) forFormula (II), either R^(10a) or R^(12a) for Formula (Ij), either R^(11a)or R^(12a) for Formula (Ik) is halogen, and the other substituent in thepairing (ex. if R^(9a) is F in Formula (If), then R^(10a) is selectedfrom the following substituent listing), is a member selected from NH₂,N(CH₃)H, and N(CH₃)₂.

In another exemplary embodiment, the compound has a structure which is amember selected from:

in which R* and R** are members selected from: H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. In an exemplary embodiment, the compound is amember selected from

wherein R^(1a) is a member selected from a negative charge, H and a saltcounterion.

In another exemplary embodiment, the compound has a structure which is amember selected from:

wherein q is 1 and R^(g) is a member selected from fluoro, chloro andbromo.

In another exemplary embodiment, the compounds and embodiments describedabove in Formulae (I)-(Io) can form a hydrate with water, a solvate withan alcohol (e.g. methanol, ethanol, propanol); an adduct with an aminocompound (e.g. ammonia, methylamine, ethylamine); an adduct with an acid(e.g. formic acid, acetic acid); complexes with ethanolamine, quinoline,amino acids, and the like.

In another exemplary embodiment, the compound has a structure accordingto Formula (Ip):

in which R^(x2) is a member selected from substituted or unsubstitutedC₁-C₅ alkyl and substituted or unsubstituted C₁-C₅ heteroalkyl. R^(y2)and R^(z2) are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

In another exemplary embodiment, the compound has a structure accordingto Formula (Iq):

wherein B is boron. R^(x2) is a member selected from substituted orunsubstituted C₁-C₅ alkyl and substituted or unsubstituted C₁-C₅heteroalkyl. R^(y2) and R^(z2) are members independently selected fromH, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. In another exemplaryembodiment, at least one member selected from R^(3a), R^(4a), R^(5a),R^(6a), R^(7a), R^(8a), R^(9a), R^(10a), R^(11a) and R^(12a) is a memberselected from nitro, cyano and halogen.

In another exemplary embodiment, the compound has a structure which is amember selected from the following Formulae:

In another exemplary embodiment, the compound has a formula according toFormulae (Ib)-(Ie) wherein at least one member selected from R^(3a),R^(4a), R^(5a), R^(6a), R^(7a), R^(8a), R^(9a), R^(10a), R^(11a) andR^(12a) is a member selected from nitro, cyano, fluoro, chloro, bromoand cyanophenoxy. In another exemplary embodiment, the compound is amember selected from

In another exemplary embodiment, the compound is a member selected from

In another exemplary embodiment, there is a proviso that the compoundcannot have a structure according to Formula (Iaa):

wherein R^(6b), R^(9b), R^(10b), R^(11b) and R^(12b) have the samesubstituent listings as described for Formulae (Ix) and (Iy) above.

In another exemplary embodiment, the invention provides poly- ormulti-valent species of the compounds of the invention. In an exemplaryembodiment, the invention provides a dimer of the compounds describedherein. In an exemplary embodiment, the invention provides a dimer ofthe compounds described herein. In an exemplary embodiment, theinvention provides a dimer of a compound which is a member selected fromC1-C96. In an exemplary embodiment the dimer is a member selected from

In an exemplary embodiment, the invention provides an anhydride of thecompounds described herein. In an exemplary embodiment, the inventionprovides an anhydride of the compounds described herein. In an exemplaryembodiment, the invention provides an anhydride of a compound which is amember selected from C1-C96. In an exemplary embodiment the anhydride isa member selected from

In an exemplary embodiment, the invention provides a trimer of thecompounds described herein. In an exemplary embodiment, the inventionprovides a trimer of the compounds described herein. In an exemplaryembodiment, the invention provides a trimer of a compound which is amember selected from C1-C96. In an exemplary embodiment the trimer is amember selected from

In an exemplary embodiment, the compound has a structure which is a

In an exemplary embodiment, the compound has a structure according toFormula (III):

I. b.) Cyclic Borinic Esters

In one aspect, the invention provides compounds useful in the methodswhich have a structure according to Formula VII:

wherein the variables R^(1a), A, D, E, G, J, W and M are describedelsewhere herein.

In an exemplary embodiment of Formula (VII), R¹ is substituted orunsubstituted alkyl (C₁-C₄). In an exemplary embodiment of Formula(VII), R¹ is substituted or unsubstituted alkyloxy. In an exemplaryembodiment of Formula (VII), R¹ is substituted or unsubstitutedcycloalkyl (C₃-C₇). In an exemplary embodiment of Formula (VII), R¹ issubstituted or unsubstituted alkenyl. In a further exemplary embodimentthereof, the substituted alkenyl has the structure

wherein R²³, R²⁴, and R² are each members independently selected from H,haloalkyl, aralkyl, substituted aralkyl, (CH₂)_(r)OH (where r=1 to 3),CH₂NR²⁶R²⁷ (wherein R²⁶ and R²⁷ are independently selected from hydrogenand alkyl), CO₂H, CO₂ alkyl, CONH₂, S-alkyl, S-aryl, SO₂ alkyl, SO₃H,SCF₃, CN, halogen, CF₃, NO₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl.

In another exemplary embodiment of Formula (VII), R¹ is a substituted orunsubstituted alkynyl. In a further exemplary embodiment thereof, thesubstituted alkynyl has the structure

wherein R²³ is defined as before.

In an exemplary embodiment of Formula (VII), R¹ is substituted orunsubstituted aryl. In a further exemplary embodiment thereof thesubstituted aryl has the structure

wherein R²⁸, R²⁹, R³⁰, R³¹ and R³² are each members independentlyselected from H, aralkyl, substituted aralkyl, (CH₂)_(s)OH (where s=1 to3), CO₂H, CO₂ alkyl, CONH₂, CONHalkyl, CON(alkyl)₂, OH, alkoxy, aryloxy,SH, S-alkyl, S-aryl, SO₂ alkyl, SO₃H, SCF₃, CN, halogen, CF₃, NO₂,(CH₂)_(t)NR²⁶R²⁷ (wherein R²⁶ and R²⁷ are independently selected fromhydrogen, alkyl, and alkanoyl) (t=0 to 2), SO₂NH₂, OCH₂CH₂NH₂,OCH₂CH₂NHalkyl, OCH₂CH₂N(alkyl)₂, oxazolidin-2-yl, alkyl substitutedoxazolidin-2-yl, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

In an exemplary embodiment of Formula (VII), R¹ is a substituted orunsubstituted aralkyl. In a further exemplary embodiment thereof thesubstituted aralkyl has the structure

wherein R²⁸, R²⁹, R³⁰, R³¹ and R³² are defined as before, and n1 is aninteger selected from 1 to 15.

In an exemplary embodiment of Formula (VII), R¹ is a substituted orunsubstituted heteroaryl. In a further exemplary embodiment thereof,heteroaryl has the structure

wherein X is a member selected from CH═CH, N═CH, NR³⁵ (wherein R³⁵═H,alkyl, aryl or benzyl), O, or S. Y═CH or N. R³³ and R³⁴ are each membersindependently selected from H, haloalkyl, aralkyl, substituted aralkyl,(CH₂)_(u)OH (where u=1, 2 or 3), (CH₂)_(v)NR²⁶R²⁷ (wherein R²⁶ and R²⁷are independently selected from hydrogen, alkyl and alkanoyl) (v=0 to3), CO₂H, CO₂ alkyl, CONH₂, S-alkyl, S-aryl, SO₂ alkyl, SO₃H, SCF₃, CN,halogen, CF₃, NO₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

The structures of the invention also permit solvent interactions thatmay afford structures (Formula VIIg) that include atoms derived from thesolvent encountered by the compounds of the invention during syntheticmanipulations and therapeutic uses. Structure VIIg arises from theformation of a dative bond between the solvent(s) with the Lewis acidicboron center. Thus, such solvent complexes could be stable entities withcomparative bioactivities. Such structures are expressly contemplated bythe present invention where R*** is H or alkyl.

In an exemplary embodiment, the compound has a structure which is amember selected from 2-(3-Chlorophenyl)-[1,3,2]-dioxaborolane,(3-Chlorophenyl)(4′-fluoro-(2′-(methoxymethoxy)-methyl)-phenyl)-borinicacid, 1-(3-Chlorophenyl)-5-fluoro-1,3-dihydrobenzo[c][1,2]oxaborole,1-(3-Chlorophenyl)-6-fluoro-1,3-dihydrobenzo[c][1,2]oxaborole,1-(3-Chlorophenyl)-1,3-dihydrobenzo[c][1,2]oxaborole,5-Chloro-1-(3-Fluorophenyl)-1,3-dihydrobenzo[c][1,2]oxaborole,2-(3-fluorophenyl)-[1,3,2]-dioxaborolane,3-(Benzo[c][1,2]oxaborol-1(3H)-yl)benzonitrile,2-(3-cyanophenyl)-[1,3,2]-dioxaborolane,(3-Chlorophenyl)(5′-fluoro-(2′-(methoxymethoxy)methyl)-phenyl)-borinicacid, 1-(3-Chlorophenyl)-1,3-dihydro-3,3-dimethylbenzo[c][1,2]oxaborole,(3-Chlorophenyl)(2-(2-(methoxymethoxy)propan-2-yl)phenylborinic acid,1-(3-Chlorophenyl)-1,3-dihydro-3,3-dimethylbenzo[c][1,2]oxaborole,1-(4-Chlorophenyl)-1,3-dihydrobenzo[c][1,2]oxaborole,2-(4-chlorophenyl)-[1,3,2]-dioxaborolane,4-(Benzo[c][1,2]oxaborol-1(3H)-yl)benzonitrile,2-(4-cyanophenyl)-[1,3,2]-dioxaborolane,4-(5-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)benzonitrile,2-(4-cyanophenyl)-[1,3,2]-dioxaborolane,3-(5-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)benzonitrile,2-(3-cyanophenyl)-[1,3,2]-dioxaborolane,3-(6-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)benzonitrile,2-(3-cyanophenyl)-[1,3,2]-dioxaborolane,1-(3-Cyanophenyl)-5,6-dimethoxy-1,3-dihydrobenzo[c][1,2]-oxaborole,2-(3-chlorophenyl)-[1,3,2]-dioxaborolane,(4-(5-(Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenylmethanamine,5-Fluoro-2-(methoxymethoxymethyl)phenyl]-[1,3,2]-dioxaborolane,4-(5-(Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenylmethanamine,(3-(5-(Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)-phenylmethanamine,(4-(5-(Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,(3-(5-(Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,3-(6-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenol,3-(5-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)pyridine,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,2-[(Methoxymethoxy)methyl]phenyl boronic acid,2-[(Methoxymethoxymethyl)phenyl]-[1,3,2]-dioxaborolane,Bis[2-(methoxymethoxymethyl)phenyl]borinic acid,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)phenyl)-N,N-dimethylmethanamine,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)-5-chlorophenyl)-N,N-dimethylmethanamine,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)-5-chlorophenyl)methanol,(2-(Benzo[c][1,2]oxaborol-1(3H)-yl)-5-chlorophenyl)methanol,(5-Chloro-2-(5-chlorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,Bis[4-chloro-2-(methoxymethoxymethyl)phenyl]borinic acid,(5-Chloro-2-(5-chlorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methanol,(5-Chloro-2-(5-chlorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl-N,N-dimethylmethanamine,1-(4-chloro-2-methoxyphenyl)-1,3-dihydrobenzo[c][1,2]benzoxaborole,4-Chloro-2-methoxyphenylboronic acid ethylene glycol ester,1-(4-chloro-2-methoxyphenyl)-1,3-dihydrobenzo[c][1,2]benzoxaborole,2-(Benzo[c][1,2]oxaboral-1(3H)-yl)-5-chlorophenol,2-(3-(Benzo[c][1.2]oxaborol-1(3H)-yl)phenoxy)-5-chlorophenol,2-(3-(Benzo[c][1,2]oxaborol-1(3H)-yl)Phenoxy)-5-chlorophenol4-((3-(5-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl)phenyl)methyl)morpholine,3-(5-Fluorobenzo[c][1,2]oxaborol-1(3H)-yl]phenyl)-methyl8-hydroxy-quinoline-2-carboxylate,1-(3-Chlorophenyl)-2,3-dihydro-2-(methoxymethy)-1H-benzo[c][1,2]azaborole,3-Chlorophenyl 2-[N,N-bis(methoxymethyl)aminomethyl]phenylborinic acid,1-(3-Chlorophenyl)-2,3-dihydro-2-(methoxymethy)-1H-benzo[c][1,2]azaborole,1-(3-Chlorophenyl)-1,3,4,5-tetrahydrobenzo-[c][1,2]-oxaborepine,1-(3-Chlorophenyl)-1,3,4,5-tetrahydrobenzo[c][1,2]oxaborepine,1-(3-Chlorophenyl)-3,4-dihydro-1H-benzo[c][1,2]-oxaborinine,2-(3-Chlorophenyl)-[1,3,2]dioxaborolane,(3-Chlorophenyl)(2′-(2-(methoxymethoxy)ethyl)phenyl)borinic acid, and1-(3-Chlorophenyl)-3,4-dihydro-1H-benzo[c][1,2]oxaborinine.

I. c.) 2′-Amino Ribofuranoses

In another aspect, the invention provides compounds useful in themethods which is a 2′-amino ribofuranose. In an exemplary embodiment,the 1′-position of the ribofuranose is substituted with a memberselected from substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. In another exemplary embodiment, the1′-position of the ribofuranose is substituted with a member selectedfrom substituted or unsubstituted purine, substituted or unsubstitutedpyrimidine, substituted or unsubstituted pyridine and substituted orunsubstituted imidazole. In another exemplary embodiment, the1′-position of the ribofuranose is substituted with a member selectedfrom substituted or unsubstituted nicotinic acid, substituted orunsubstituted nicotinamide, substituted or unsubstituted nucleic acidbase, substituted or unsubstituted adenine,

substituted or unsubstituted cytosine, substituted or unsubstitutedguanine, substituted or unsubstituted thymine, substituted orunsubstituted uracil, substituted or unsubstituted N,N-dimethyl guanine,substituted or unsubstituted dihydrouracil, substituted or unsubstituted4-thiouridine and substituted or unsubstituted inosine. In anotherexemplary embodiment, the compound has a structure according to Formula(VIII):

wherein L is a member selected from substituted or unsubstituted purine,substituted or unsubstituted pyrimidine, substituted or unsubstitutedpyridine and substituted or unsubstituted imidazole. M, as definedherein earlier, is a member selected from O, S, and NR². R⁴⁰ and R⁴¹ areeach members independently selected from H, aralkyl, substitutedaralkyl, (CH₂)_(s)OH (where s=1 to 3), CO₂H, CO₂ alkyl, C(O)NH₂,C(O)NHalkyl, CON(alkyl)₂, C(O)R²³, OH, alkoxy, aryloxy, SH, S-alkyl,S-aryl, SO₂ alkyl, SO₃H, SCF₃, CN, halogen, CF₃, NO₂, (CH₂)_(t)NR²⁶R²⁷(wherein R²⁶ and R²⁷ are independently selected from hydrogen, alkyl,and alkanoyl) (t=0 to 2), SO₂NH₂, OCH₂CH₂NH₂, OCH₂CH₂NHalkyl,OCH₂CH₂N(alkyl)₂, oxazolidin-2-yl, alkyl substituted oxazolidin-2-yl,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl,

R⁴³, R⁴⁴, and R⁴⁵ are each members independently selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. R⁴³ and R⁴⁴, together with theatoms to which they are attached, are optionally joined to form a 4 to 7membered ring. R⁴³ and R⁴⁵, together with the atoms to which they areattached, are optionally joined to form a 4 to 7 membered ring. R⁴⁴ andR⁴⁵, together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring. A, D, E and G are all definedelsewhere herein. Z is a member selected from CR⁴⁶ and N. Thecombinations of nitrogens (A+D+E+G+Z) is an integer selected from 0 to4. At least two members selected from R⁹, R¹⁰, R¹¹, R¹² and R⁴⁶,together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring.

In an exemplary embodiment,

is a member selected from:

In another exemplary embodiment, the compound has a formula according tothe following formulae:

In an exemplary embodiment, the compound is a member selected from:

I. d.) 3′-Amino Ribofuranoses

In another aspect, the invention provides compounds useful in themethods which is a 3′-amino ribofuranose. In an exemplary embodiment,the 1′-position of the ribofuranose is substituted with a memberselected from substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. In another exemplary embodiment, the1′-position of the ribofuranose is substituted with a member selectedfrom substituted or unsubstituted purine, substituted or unsubstitutedpyrimidine, substituted or unsubstituted pyridine, and substituted orunsubstituted imidazole. In another exemplary embodiment, the1′-position of the ribofuranose is substituted with a member selectedfrom substituted or unsubstituted nicotinic acid, substituted orunsubstituted nicotinamide, substituted or unsubstituted nucleic acidbase, substituted or unsubstituted adenine,

substituted or unsubstituted cytosine, substituted or unsubstitutedguanine, substituted or unsubstituted thymine, substituted orunsubstituted uracil, substituted or unsubstituted N,N-dimethyl guanine,substituted or unsubstituted dihydrouracil, substituted or unsubstituted4-thiouridine and substituted or unsubstituted inosine. In anotherexemplary embodiment, the compound has a structure according to Formula(VIIIc):

wherein L is a member selected from substituted or unsubstituted purine,substituted or unsubstituted pyrimidine, substituted or unsubstitutedpyridine and substituted or unsubstituted imidazole. M, as definedherein earlier, is a member selected from O, S, and NR². R⁴⁰ and R⁴¹ areeach members independently selected from H, aralkyl, substitutedaralkyl, (CH₂)_(s)OH (where s=1 to 3), CO₂H, CO₂ alkyl, C(O)NH₂,C(O)NHalkyl, CON(alkyl)₂, C(O)R²³, OH, alkoxy, aryloxy, SH, S-alkyl,S-aryl, SO₂ alkyl, SO₃H, SCF₃, CN, halogen, CF₃, NO₂, (CH₂)_(t)NR²⁶R²⁷(wherein R²⁶ and R²⁷ are independently selected from hydrogen, alkyl,and alkanoyl) (t=0 to 2), SO₂NH₂, OCH₂CH₂NH₂, OCH₂CH₂NHalkyl,OCH₂CH₂N(alkyl)₂, oxazolidin-2-yl, alkyl substituted oxazolidin-2-yl,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl,

R⁴³, R⁴⁴, and R⁴⁵ are each members independently selected fromsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. R⁴³ and R⁴⁴, together with theatoms to which they are attached, are optionally joined to form a 4 to 7membered ring. R⁴³ and R⁴⁵, together with the atoms to which they areattached, are optionally joined to form a 4 to 7 membered ring. R⁴⁴ andR⁴⁵, together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring. A, D, E and G are all definedelsewhere herein. Z is a member selected from CR⁴⁶ and N. Thecombinations of nitrogens (A+D+E+G+Z) is an integer selected from 0 to4. At least two members selected from R⁹, R¹⁰, R¹¹, R¹² and R⁴⁶,together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring.

In an exemplary embodiment,

is a member selected from:

In another exemplary embodiment, the compound has a formula according

In an exemplary embodiment, the compound is a member selected from:

I. e.) Acyclic Boronic Acids and Esters, Part I

Acyclic boronic acids and esters such as those described in this sectioncan also be utilized in the invention. These compounds can be used tokill or inhibit the growth of the microorganisms described herein, aswell as treat the diseases described herein. In addition, thesecompounds can be used as synthetic intermediates in the generation ofthe compounds described herein.

In another aspect, the compound has a structure according to thefollowing formula:

in which R¹ and R² are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. R¹ and R², together with theatoms to which they are attached, can be optionally joined to form a 4-to 7-membered ring. Z1 is a member selected from

wherein each R^(3a) and R^(4a) is a member independently selected fromH, cyano, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. R⁵ is amember selected from halogen and OR⁶. R⁶ is a member selected from H,cyano, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. A is a member selected fromCR^(9a) and N. D is a member selected from CR^(10a) and N. E is a memberselected from CR^(11a) and N. G is a member selected from CR^(12a) andN. R^(9a), R^(10a), R^(11a) and R^(12a) are members independentlyselected from H, OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*, —S(O)₂NR*R**,—C(O)R*, —C(O)OR*, —C(O)NR*R**, nitro, halogen, cyano, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. Each R* and R** is a member independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. R^(9a)and R^(10a), along with the atoms to which they are attached, areoptionally joined to form a ring. R^(10a) and R^(11a), along with theatoms to which they are attached, are optionally joined to form a ring.R^(11a) and R^(12a), along with the atoms to which they are attached,are optionally joined to form a ring. The combination of nitrogens(A+D+E+G) is an integer selected from 0 to 3.

In an exemplary embodiment, there is a proviso that the compound is nota member selected from:

In an exemplary embodiment, the compound has a structure according toFormula IXa

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberindependently selected from H, cyano, substituted or unsubstitutedmethyl, substituted or unsubstituted ethyl, trifluoromethyl, substitutedor unsubstituted hydroxymethyl, substituted or unsubstitutedhydroxyalkyl, substituted or unsubstituted benzyl, substituted orunsubstituted phenyl, substituted or unsubstituted mercaptomethyl,substituted or unsubstituted mercaptoalkyl, substituted or unsubstitutedaminomethyl, substituted or unsubstituted alkylaminomethyl, substitutedor unsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted indolyl and substituted orunsubstituted amido. In another exemplary embodiment, each R^(3a) andR^(4a) is a member independently selected from cyano, substituted orunsubstituted methyl, substituted or unsubstituted ethyl,trifluoromethyl, substituted or unsubstituted hydroxymethyl, substitutedor unsubstituted hydroxyalkyl, substituted or unsubstituted benzyl,substituted or unsubstituted phenyl, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted indolyl, substituted or unsubstituted amido.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. In another exemplary embodiment,R^(3a) and R^(4a) is a member selected from methyl, ethyl, propyl,isopropyl, butyl, t-butyl, phenyl and benzyl. In another exemplaryembodiment, R^(3a) is H and R^(4a) is a member selected from methyl,ethyl, propyl, isopropyl, butyl, t-butyl, phenyl and benzyl. In anotherexemplary embodiment, R^(3a) is H and R^(4a)H.

In another exemplary embodiment, Z1 is CHO. In another exemplaryembodiment, Z1 is

wherein R⁵ is a member selected from OH, substituted or unsubstitutedmethoxy, substituted or unsubstituted ethoxy, substituted orunsubstituted methoxymethoxy, substituted or unsubstituted ethoxyethoxy,substituted or unsubstituted trialkylsialyl, and substituted orunsubstituted tetrahydro-2H-pyran-2-yloxy. In another exemplaryembodiment, R⁵ is substituted or unsubstituted trialkylsialyl, whereinsaid trialkylsialyl is a member selected from substituted orunsubstituted trimethylsilyl, substituted or unsubstitutedtert-butyldimethylsilyl, and substituted or unsubstituted tributylsilyl.In another exemplary embodiment, R⁵ is substituted or unsubstitutedmethoxy, substituted or unsubstituted ethoxy, substituted orunsubstituted methoxymethoxy, substituted or unsubstituted ethoxyethoxy,and substituted or unsubstituted tetrahydro-2H-pyran-2-yloxy. In anotherexemplary embodiment, R⁵ is a member selected from methoxy, ethoxy,methoxymethoxy, ethoxyethoxy and tetrahydro-2H-pyran-2-yloxy. In anotherexemplary embodiment, Z1 is

In an exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) is amember independently selected from H, OR*, NR*R**, SR*, —S(O)R*,—S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, halogen, cyano,nitro, substituted or unsubstituted methoxy, substituted orunsubstituted methyl, substituted or unsubstituted ethoxy, substitutedor unsubstituted ethyl, trifluoromethyl, substituted or unsubstitutedhydroxymethyl, substituted or unsubstituted hydroxyalkyl, substituted orunsubstituted benzyl, substituted or unsubstituted phenyl, substitutedor unsubstituted phenyloxy, substituted or unsubstituted phenyl methoxy,substituted or unsubstituted thiophenyloxy, substituted or unsubstitutedpyridinyloxy, substituted or unsubstituted pyrimidinyloxy, substitutedor unsubstituted benzylfuran, substituted or unsubstituted methylthio,substituted or unsubstituted mercaptomethyl, substituted orunsubstituted mercaptoalkyl, substituted or unsubstituted phenylthio,substituted or unsubstituted thiophenylthio, substituted orunsubstituted phenyl methylthio, substituted or unsubstitutedpyridinylthio, substituted or unsubstituted pyrimidinylthio, substitutedor unsubstituted benzylthiofuranyl, substituted or unsubstitutedphenylsulfonyl, substituted or unsubstituted benzylsulfonyl, substitutedor unsubstituted phenylmethylsulfonyl, substituted or unsubstitutedthiophenylsulfonyl, substituted or unsubstituted pyridinylsulfonyl,substituted or unsubstituted pyrimidinylsulfonyl, substituted orunsubstituted sulfonamidyl, substituted or unsubstituted phenylsulfinyl,substituted or unsubstituted benzylsulfinyl, substituted orunsubstituted phenylmethylsulfinyl, substituted or unsubstitutedthiophenylsulfinyl, substituted or unsubstituted pyridinylsulfinyl,substituted or unsubstituted pyrimidinylsulfinyl, substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedtrifluoromethylamino, substituted or unsubstituted aminomethyl,substituted or unsubstituted alkylaminomethyl, substituted orunsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted benzylamino, substitutedor unsubstituted phenylamino, substituted or unsubstitutedthiophenylamino, substituted or unsubstituted pyridinylamino,substituted or unsubstituted pyrimidinylamino, substituted orunsubstituted indolyl, substituted or unsubstituted morpholino,substituted or unsubstituted alkylamido, substituted or unsubstitutedarylamido, substituted or unsubstituted ureido, substituted orunsubstituted carbamoyl, and substituted or unsubstituted piperizinyl.In an exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) areselected from the previous list of substituents with the exception of—C(O)R*, —C(O)OR*, —C(O)NR*R**.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy,4-fluorobenzyloxy, unsubstituted phenyl, unsubstituted benzyl. In anexemplary embodiment, R^(9a) is H and R^(12a) is H. In an exemplaryembodiment, the compound has a substitutent combination for R^(9a),R^(10a) R^(11a), and R^(12a) which is a member selected from thosedescribed in Formulae (I), (Ia) (Ib), (Ic), (Id), (Ie), (If), (Ig),(Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip), (Iq), (Ir), (Is),(It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae), (Iaf),(Iag), (Iah), (Iai), (Iaj), (Iak), above, and/or the subsequentparagraphs describing Formulae (I), (Ia) (Ib), (Ic), (Id), (Ie), (If),(Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip), (Iq), (Ir),(Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae),(Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak).

In an exemplary embodiment, the compound is an acyclic boronic acid or

ester in which a portion of the acyclic boronic acid or ester as inFigure (IXb) below

is a member selected from a structure in FIG. 12. In another exemplaryembodiment, the compound is a dimer, anhydride or trimer of an acyclicboronic acid or ester described herein. In another exemplary embodiment,the compound is a dimer, anhydride or trimer of an acyclic boronic acidor ester in which a portion of the acyclic boronic acid or ester as inFigure (IXb) is a member selected a structure in FIG. 12.

In an exemplary embodiment, R¹ and R² are each members independentlyselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. R¹ and R², together with the atomsto which they are joined, can optionally form a member selected fromsubstituted or unsubstituted dioxaborolane, substituted or unsubstituteddioxaborinane, substituted or unsubstituted dioxaborepane.

In an exemplary embodiment, R¹ and R² are each members independentlyselected from H, methyl, ethyl, propyl, isopropyl, butyl, t-butyl,phenyl and benzyl. In an exemplary embodiment, R¹ and R² are eachmembers independently selected from H, methyl, isopropyl, and phenyl. Inan exemplary embodiment, R¹ and R² are methyl. In an exemplaryembodiment, R¹ and R² are isopropyl. In an exemplary embodiment, R¹ andR² are H.

In another exemplary embodiment, R¹ and R², together with the atoms towhich they are joined, form a member selected from substituted orunsubstituted dioxaborolane, substituted or unsubstituted dioxaborinane,substituted or unsubstituted dioxaborepane. In another exemplaryembodiment, R¹ and R², together with the atoms to which they are joined,form a member selected from dioxaborolane, substituted or unsubstitutedtetramethyldioxaborolane, substituted or unsubstitutedphenyldioxaborolane, dioxaborinane, dimethyldioxaborinane anddioxaborepane.

In an exemplary embodiment, the compound is a member selected from

In an exemplary embodiment, the compound is a member selected from:

In an exemplary embodiment, the compound is a member selected from

In another exemplary embodiment, the compounds and embodiments describedherein can form a hydrate with water, a solvate with an alcohol (e.g.methanol, ethanol, propanol); an adduct with an amino compound (e.g.ammonia, methylamine, ethylamine); an adduct with an acid (e.g. formicacid, acetic acid); complexes with ethanolamine, quinoline, amino acids,and the like.

In an exemplary embodiment, acyclic boronic esters described herein canbe used as intermediates in the synthesis of the compounds describedherein. In another exemplary embodiment, the acyclic boronic estersdescribed herein can be used as intermediates in the synthesis of acompound which is a member selected from Formulae (I), (Ia) (Ib), (Ic),(Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io),(Ip), (Iq), (Ir), (Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab),(Iac), (Iad), (Iae), (Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak).

I. f) Acyclic Boronic Acids and Esters, Part II

Acyclic boronic acids and esters described herein can also be utilizedin the invention. These compounds can be used to kill or inhibit thegrowth of the microorganisms described herein, as well as treat thediseases described herein. In addition, these compounds can be used assynthetic intermediates in the generation of other compounds describedherein. In an exemplary embodiment, these other compounds are the cyclicboronic esters and cyclic borinic esters described herein.

In another aspect, the compound has a structure according to thefollowing formula:

in which R¹ and R² are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. R¹ and R², together with theatoms to which they are attached, can be optionally joined to form a 4-to 7-membered ring. X is a member selected from substituted orunsubstituted triflate, halogen, substituted or unsubstituted sulfonicesters and substituted or unsubstituted acyloxy groups, and substitutedor unsubstituted diazo. R^(3a) and R^(4a) are members independentlyselected from H, cyano, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. A is amember selected from CR^(9a) and N. D is a member selected from CR^(10a)and N. E is a member selected from CR^(11a) and N. G is a memberselected from CR^(12a) and N. R^(9a), R^(10a), R^(11a) and R^(12a) aremembers independently selected from H, OR*, NR*R**, SR*, —S(O)R*,—S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, nitro, halogen,cyano, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. Each R* and R** is a memberindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl.R^(9a) and R^(10a), along with the atoms to which they are attached, areoptionally joined to form a ring. R^(10a) and R^(11a), along with theatoms to which they are attached, are optionally joined to form a ring.R^(11a) and R^(12a), along with the atoms to which they are attached,are optionally joined to form a ring. The combination of nitrogens(A+D+E+G) is an integer selected from 0 to 3.

In an exemplary embodiment, this aspect has the proviso that thecompound is not:

In an exemplary embodiment, the compound has a structure according toFormula (Xa)

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberindependently selected from H, cyano, substituted or unsubstitutedmethyl, substituted or unsubstituted ethyl, trifluoromethyl, substitutedor unsubstituted hydroxymethyl, substituted or unsubstitutedhydroxyalkyl, substituted or unsubstituted benzyl, substituted orunsubstituted phenyl, substituted or unsubstituted mercaptomethyl,substituted or unsubstituted mercaptoalkyl, substituted or unsubstitutedaminomethyl, substituted or unsubstituted alkylaminomethyl, substitutedor unsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted indolyl and substituted orunsubstituted amido. In another exemplary embodiment, each R^(3a) andR^(4a) is a member independently selected from cyano, substituted orunsubstituted methyl, substituted or unsubstituted ethyl,trifluoromethyl, substituted or unsubstituted hydroxymethyl, substitutedor unsubstituted hydroxyalkyl, substituted or unsubstituted benzyl,substituted or unsubstituted phenyl, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted indolyl, substituted or unsubstituted amido.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. In another exemplary embodiment,R^(3a) and R^(4a) is a member selected from methyl, ethyl, propyl,isopropyl, butyl, t-butyl, phenyl and benzyl. In another exemplaryembodiment, R^(3a) is H and R^(4a) is a member selected from methyl,ethyl, propyl, isopropyl, butyl, t-butyl, phenyl and benzyl. In anotherexemplary embodiment, R^(3a) is H and R^(4a)H.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) isa member selected from H, OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*,—S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, halogen, cyano, nitro,substituted or unsubstituted methoxy, substituted or unsubstitutedmethyl, substituted or unsubstituted ethoxy, substituted orunsubstituted ethyl, trifluoromethyl, substituted or unsubstitutedhydroxymethyl, substituted or unsubstituted hydroxyalkyl, substituted orunsubstituted benzyl, substituted or unsubstituted phenyl, substitutedor unsubstituted phenyloxy, substituted or unsubstituted phenyl methoxy,substituted or unsubstituted thiophenyloxy, substituted or unsubstitutedpyridinyloxy, substituted or unsubstituted pyrimidinyloxy, substitutedor unsubstituted benzylfuran, substituted or unsubstituted methylthio,substituted or unsubstituted mercaptomethyl, substituted orunsubstituted mercaptoalkyl, substituted or unsubstituted phenylthio,substituted or unsubstituted thiophenylthio, substituted orunsubstituted phenyl methylthio, substituted or unsubstitutedpyridinylthio, substituted or unsubstituted pyrimidinylthio, substitutedor unsubstituted benzylthiofuranyl, substituted or unsubstitutedphenylsulfonyl, substituted or unsubstituted benzylsulfonyl, substitutedor unsubstituted phenylmethylsulfonyl, substituted or unsubstitutedthiophenylsulfonyl, substituted or unsubstituted pyridinylsulfonyl,substituted or unsubstituted pyrimidinylsulfonyl, substituted orunsubstituted sulfonamidyl, substituted or unsubstituted phenylsulfinyl,substituted or unsubstituted benzylsulfinyl, substituted orunsubstituted phenylmethylsulfinyl, substituted or unsubstitutedthiophenylsulfinyl, substituted or unsubstituted pyridinylsulfinyl,substituted or unsubstituted pyrimidinylsulfinyl, substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedtrifluoromethylamino, substituted or unsubstituted aminomethyl,substituted or unsubstituted alkylaminomethyl, substituted orunsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted benzylamino, substitutedor unsubstituted phenylamino, substituted or unsubstitutedthiophenylamino, substituted or unsubstituted pyridinylamino,substituted or unsubstituted pyrimidinylamino, substituted orunsubstituted indolyl, substituted or unsubstituted morpholino,substituted or unsubstituted alkylamido, substituted or unsubstitutedarylamido, substituted or unsubstituted ureido, substituted orunsubstituted carbamoyl, and substituted or unsubstituted piperizinyl.In an exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) areselected from the previous list of substituents with the exception of—C(O)R*, —C(O)OR*, —C(O)NR*R**.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy,4-fluorobenzyloxy, unsubstituted phenyl, unsubstituted benzyl. In anexemplary embodiment, R^(9a) is H and R^(12a) is H. In an exemplaryembodiment, the compound has a substitutent combination for R^(9a),R^(10a) R^(11a), and R^(12a) which is a member selected from thosedescribed in Formulae (I), (Ia) (Ib), (Ic), (Id), (Ie), (If), (Ig),(Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip), (Iq), (Ir), (Is),(It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae), (Iaf),(Iag), (Iah), (Iai), (Iaj), (Iak), above, and/or the subsequentparagraphs describing Formulae (I), (Ia) (Ib), (Ic), (Id), (Ie), (If),(Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip), (Iq), (Ir),(Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae),(Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak).

In an exemplary embodiment, the compound is an acyclic boronic acid orester in which a portion of the acyclic boronic acid or ester is as inFigure (IXb) below

is a member selected from a structure in FIG. 12. In another exemplaryembodiment, the compound is a dimer, anhydride or trimer of an acyclicboronic acid or ester described herein. In another exemplary embodiment,the compound is a dimer, anhydride or trimer of an acyclic boronic acidor ester in which a portion of the acyclic boronic acid or ester as inFigure (IXb) is a member selected a structure in FIG. 12.

In an exemplary embodiment, R¹ and R² are each members independentlyselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. R¹ and R², together with the atomsto which they are joined, can optionally form a member selected fromsubstituted or unsubstituted dioxaborolane, substituted or unsubstituteddioxaborinane, substituted or unsubstituted dioxaborepane.

In an exemplary embodiment, X is a member selected from triflate,chloro, bromo, iodo, substituted or unsubstituted sulfonic esters,substituted or unsubstituted acyloxy groups, and substituted orunsubstituted diazo. In an exemplary embodiment, X is a sulfonic estergroup, which is a member selected from substituted or unsubstitutedmesylate, substituted or unsubstituted tosylate, substituted orunsubstituted brosylate and substituted or unsubstituted nosylate. In anexemplary embodiment, X is an acyloxy group, which is a member selectedfrom substituted or unsubstituted acetoxy and substituted orunsubstituted trifluoroacetoxy. In another exemplary embodiment, X is amember selected from bromo, iodo, mesylate and diazo. In anotherexemplary embodiment, X is a member selected from bromo and iodo.

In another exemplary embodiment, R¹ and R², together with the atoms towhich they are joined, form a member selected from dioxaborolane,substituted or unsubstituted tetramethyldioxaborolane, substituted orunsubstituted phenyldioxaborolane, dioxaborinane, dimethyldioxaborinaneand dioxaborepane.

In another exemplary embodiment, R^(3a) and R^(4a) are each membersindependently selected from H, methyl, ethyl, propyl, butyl, phenyl,benzyl, cyano, halogen and nitro.

In an exemplary embodiment, the compound is a member selected from

In an exemplary embodiment, the compound is a member selected from

In an exemplary embodiment, acyclic boronic esters described herein canbe used as intermediates in the synthesis of the compounds describedherein. In another exemplary embodiment, the acyclic boronic estersdescribed herein can be used as intermediates in the synthesis of acompound which is a member selected from Formulae (I), (Ia) (Ib), (Ic),(Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io),(Ip), (Iq), (Ir), (Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab),(Iac), (Iad), (Iae), (Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak).

I. e.) Additional Compounds

Compounds such as those described herein can also be utilized in theinvention. The compounds of the invention can form between the 2′,3′diol of the ribose ring of a nucleic acid, nucleoside or nucleotide, anda cyclic or acyclic boronic ester such as those described herein. Thesecompounds can be used in a human or in an animal to kill or inhibit thegrowth of the microorganisms described herein, as well as to treat thediseases described herein. These compounds can be formed in vitro aswell as in vivo. Methods of making these compounds are provided in theExamples section.

In another aspect, the invention provides a compound having a structureaccording to the following formula:

wherein B is boron. L is a member selected from OR⁷, substituted orunsubstituted purine, substituted or unsubstituted pyrimidine,substituted or unsubstituted pyridine and substituted or unsubstitutedimidazole. R⁷ is a member selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. A is a member selected from OH,substituted or unsubstituted monophosphate, substituted or unsubstituteddiphosphate, substituted or unsubstituted triphosphate,

A1 is a nucleic acid sequence which comprises between 1 and 100nucleotides. Q is a member selected from substituted or unsubstitutedheterocycloalkyl and substituted or unsubstituted heteroaryl. Qcomprises said boron and at least one oxygen.

In an exemplary embodiment, the aspect has the proviso that the compoundcannot comprise a member selected from C1-C40.

In an exemplary embodiment, the aspect has a proviso that the compoundcannot comprise a member which is described in FIG. 11. In an exemplaryembodiment, the aspect has a proviso that the compound cannot involve acompound which is described in expired U.S. Pat. No. 5,880,188.

In an exemplary embodiment, the compound has a structure according tothe following formula (XIIa):

wherein M is a member selected from O and S. J is a member selected from(CR^(3a)R^(4a))_(n1) and CR^(5a). R^(3a), R^(4a), and R^(5a) are membersindependently selected from H, halogen, cyano, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. n1 is an integer selected from 0 to 2. W is amember selected from C═O (carbonyl), (CR^(6a)R^(7a))_(m) and CR^(5a).R^(6a), R^(7a), and Rsa are members independently selected from H,halogen, cyano, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. Theindex m1 is an integer selected from 0 and 1. A is a member selectedfrom CR^(9a) and N. D is a member selected from CR^(10a) and N. E is amember selected from CR^(11a) and N. G is a member selected fromCR^(12a) and N. R^(9a) R^(10a), R^(11a) and R^(12a) are membersindependently selected from H, OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*,—S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, nitro, halogen, cyano,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. Each R* and R** are membersindependently selected from H, nitro, halogen, cyano, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. The combination of nitrogens (A+D+E+G) is aninteger selected from 0 to 3. A member selected from R^(3a), R^(4a) andR^(5a) and a member selected from R^(6a), R^(7a) and R^(8a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring. R^(3a) and R^(4a), together with the atoms towhich they are attached, are optionally joined to form a 4 to 7 memberedring. R^(6a) and R^(7a), together with the atoms to which they areattached, are optionally joined to form a 4 to 7 membered ring. R^(9a)and R^(10a), together with the atoms to which they are attached, areoptionally joined to form a 4 to 7 membered ring. R^(10a) and R^(11a),together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring. R^(11a) and R^(12a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberindependently selected from H, cyano, substituted or unsubstitutedmethyl, substituted or unsubstituted ethyl, trifluoromethyl, substitutedor unsubstituted hydroxymethyl, substituted or unsubstitutedhydroxyalkyl, substituted or unsubstituted benzyl, substituted orunsubstituted phenyl, substituted or unsubstituted mercaptomethyl,substituted or unsubstituted mercaptoalkyl, substituted or unsubstitutedaminomethyl, substituted or unsubstituted alkylaminomethyl, substitutedor unsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted indolyl and substituted orunsubstituted amido. In another exemplary embodiment, each R^(3a) andR^(4a) is a member independently selected from cyano, substituted orunsubstituted methyl, substituted or unsubstituted ethyl,trifluoromethyl, substituted or unsubstituted hydroxymethyl, substitutedor unsubstituted hydroxyalkyl, substituted or unsubstituted benzyl,substituted or unsubstituted phenyl, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted indolyl, substituted or unsubstituted amido.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. In another exemplary embodiment,R^(3a) and R^(4a) is a member selected from methyl, ethyl, propyl,isopropyl, butyl, t-butyl, phenyl and benzyl. In another exemplaryembodiment, R^(3a) is H and R^(4a) is a member selected from methyl,ethyl, propyl, isopropyl, butyl, t-butyl, phenyl and benzyl. In anotherexemplary embodiment, R^(3a) is H and R^(4a)H.

In another exemplary embodiment, each R^(9a), R^(10a), R^(11a) andR^(12a) is a member independently selected from H, OR*, NR*R**, SR*,—S(O)R*, —S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**,halogen, cyano, nitro, substituted or unsubstituted methoxy, substitutedor unsubstituted methyl, substituted or unsubstituted ethoxy,substituted or unsubstituted ethyl, trifluoromethyl, substituted orunsubstituted hydroxymethyl, substituted or unsubstituted hydroxyalkyl,substituted or unsubstituted benzyl, substituted or unsubstitutedphenyl, substituted or unsubstituted phenyloxy, substituted orunsubstituted phenyl methoxy, substituted or unsubstitutedthiophenyloxy, substituted or unsubstituted pyridinyloxy, substituted orunsubstituted pyrimidinyloxy, substituted or unsubstituted benzylfuran,substituted or unsubstituted methylthio, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted phenylthio, substituted or unsubstitutedthiophenylthio, substituted or unsubstituted phenyl methylthio,substituted or unsubstituted pyridinylthio, substituted or unsubstitutedpyrimidinylthio, substituted or unsubstituted benzylthiofuranyl,substituted or unsubstituted phenylsulfonyl, substituted orunsubstituted benzylsulfonyl, substituted or unsubstitutedphenylmethylsulfonyl, substituted or unsubstituted thiophenylsulfonyl,substituted or unsubstituted pyridinylsulfonyl, substituted orunsubstituted pyrimidinylsulfonyl, substituted or unsubstitutedsulfonamidyl, substituted or unsubstituted phenylsulfinyl, substitutedor unsubstituted benzylsulfinyl, substituted or unsubstitutedphenylmethylsulfinyl, substituted or unsubstituted thiophenylsulfinyl,substituted or unsubstituted pyridinylsulfinyl, substituted orunsubstituted pyrimidinylsulfinyl, substituted or unsubstituted amino,substituted or unsubstituted alkylamino, substituted or unsubstituteddialkylamino, substituted or unsubstituted trifluoromethylamino,substituted or unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted benzylamino, substituted or unsubstituted phenylamino,substituted or unsubstituted thiophenylamino, substituted orunsubstituted pyridinylamino, substituted or unsubstitutedpyrimidinylamino, substituted or unsubstituted indolyl, substituted orunsubstituted morpholino, substituted or unsubstituted alkylamido,substituted or unsubstituted arylamido, substituted or unsubstitutedureido, substituted or unsubstituted carbamoyl, and substituted orunsubstituted piperizinyl. In an exemplary embodiment, R^(9a), R^(10a),R^(11a) and R^(12a) are selected from the previous list of substituentswith the exception of —C(O)R*, —C(O)OR*, —C(O)NR*R**.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy,4-fluorobenzyloxy, unsubstituted phenyl, unsubstituted benzyl.

In an exemplary embodiment, the compound has a structure according tothe following formula:

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from H, halogen, cyano, nitro,substituted or unsubstituted methoxy, substituted or unsubstitutedmethyl, substituted or unsubstituted ethoxy, substituted orunsubstituted ethyl, trifluoromethyl, substituted or unsubstitutedhydroxymethyl, substituted or unsubstituted hydroxyalkyl, substituted orunsubstituted benzyl, substituted or unsubstituted phenyl, substitutedor unsubstituted phenyloxy, substituted or unsubstituted phenyl methoxy,substituted or unsubstituted thiophenyloxy, substituted or unsubstitutedpyridinyloxy, substituted or unsubstituted pyrimidinyloxy, substitutedor unsubstituted benzylfuran, substituted or unsubstituted methylthio,substituted or unsubstituted mercaptomethyl, substituted orunsubstituted mercaptoalkyl, substituted or unsubstituted phenylthio,substituted or unsubstituted thiophenylthio, substituted orunsubstituted phenyl methylthio, substituted or unsubstitutedpyridinylthio, substituted or unsubstituted pyrimidinylthio, substitutedor unsubstituted benzylthiofuranyl, substituted or unsubstitutedphenylsulfonyl, substituted or unsubstituted benzylsulfonyl, substitutedor unsubstituted phenylmethylsulfonyl, substituted or unsubstitutedthiophenylsulfonyl, substituted or unsubstituted pyridinylsulfonyl,substituted or unsubstituted pyrimidinylsulfonyl, substituted orunsubstituted sulfonamidyl, substituted or unsubstituted phenylsulfinyl,substituted or unsubstituted benzylsulfinyl, substituted orunsubstituted phenylmethylsulfinyl, substituted or unsubstitutedthiophenylsulfinyl, substituted or unsubstituted pyridinylsulfinyl,substituted or unsubstituted pyrimidinylsulfinyl, substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedtrifluoromethylamino, substituted or unsubstituted aminomethyl,substituted or unsubstituted alkylaminomethyl, substituted orunsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted benzylamino, substitutedor unsubstituted phenylamino, substituted or unsubstitutedthiophenylamino, substituted or unsubstituted pyridinylamino,substituted or unsubstituted pyrimidinylamino, substituted orunsubstituted indolyl, substituted or unsubstituted morpholino,substituted or unsubstituted alkylamido, substituted or unsubstitutedarylamido, substituted or unsubstituted ureido, substituted orunsubstituted carbamoyl, and substituted or unsubstituted piperizinyl.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from H, fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy. In an exemplary embodiment, R^(9a) is H and R^(12a)is H. In an exemplary embodiment, the compound has a substitutentcombination for R^(9a), R^(10a), R^(11a), and R^(12a) which is a memberselected from those described in Formulae (I), (Ia) (Ib), (Ic), (Id),(Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip),(Iq), (Ir), (Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac),(Iad), (Iae), (Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak) above, and/or thesubsequent paragraphs describing Formulae (I), (Ia) (Ib), (Ic), (Id),(Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im), (In), (Io), (Ip),(Iq), (Ir), (Is), (It), (Iu), (Iv), (Iw), (Iz), (Iaa), (Iab), (Iac),(Iad), (Iae), (Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak).

In an exemplary embodiment, the portion of the cyclic boronic ester asin the figure below

is a member selected from a structure in FIG. 12.

In an exemplary embodiment, the compound has a structure according tothe following formula:

In an exemplary embodiment, the compound has a structure according tothe following formula:

In an exemplary embodiment, the compound has a structure according tothe following formula:

In another exemplary embodiment, the compound has a structure which is amember selected from Formulae (XII), (XIIa), (XIIb), (XIIc), (XIId) and(XIIe) wherein L is a member selected from substituted or unsubstitutedadenine, substituted or unsubstituted guanine, substituted orunsubstituted cytidine, substituted or unsubstituted uracil, andsubstituted or unsubstituted thymine. In another exemplary embodiment, Lis OH. In another exemplary embodiment, L is adenine.

In another exemplary embodiment, the compound has a structure which is amember selected from

In another exemplary embodiment, A1 is a nucleic acid sequence between72 and 90 nucleotides. In another exemplary embodiment, A1 is a nucleicacid sequence between 35 and 150 nucleotides. In another exemplaryembodiment, A1 is a nucleic acid sequence between 50 and 100nucleotides. In another exemplary embodiment, A1 is a nucleic acidsequence between 75 and 85 nucleotides. In another exemplary embodiment,A1 is a nucleic acid sequence which is a tRNA or a portion of a tRNA. Inanother exemplary embodiment, said tRNA or the portion of said tRNA is amember selected from alanyl tRNA, isoleucyl tRNA, leucyl tRNA, methionyltRNA, lysyl tRNA, phenylalanyl tRNA, prolyl tRNA, threonyl tRNA andvalyl tRNA. In another exemplary embodiment, said tRNA or the portion ofsaid tRNA is leucyl tRNA. In another exemplary embodiment, said tRNA orthe portion of said tRNA has a sequence which is a member selected fromSEQ ID NOS: 18-62. In another exemplary embodiment, A1 is a nucleic acidsequence wherein two final nucleotides are each cytidine.

In another exemplary embodiment, the compound further comprises a tRNAsynthetase or a portion of a tRNA synthetase which comprises the editingdomain, wherein said compound is noncovalently attached to the editingdomain of said tRNA synthetase. In another exemplary embodiment, thetRNA synthetase is a member selected from a mitochondrial tRNAsynthetase and a cytoplasmic tRNA synthetase. In another exemplaryembodiment, the tRNA synthetase is a member selected from alanyl tRNAsynthetase, isoleucyl tRNA synthetase, leucyl tRNA synthetase, methionyltRNA synthetase, lysyl tRNA synthetase, phenylalanyl tRNA synthetase,prolyl tRNA synthetase, threonyl tRNA synthetase and valyl tRNAsynthetase.

In an exemplary embodiment, the compound described herein is present ina microorganism described in this application.

In another exemplary embodiment, there is a proviso that the compound isnot present in a microorganism that is a member selected fromSaccharomyces cerevisiae, Aspergillus niger, Pseudomonas aeruginosa,Staphlococcus aureus, Aureobasidium pullulans, Fusarium solani,Penicillium pinophilum, Scopulariopsis brevicaulis, Streptoverticilliumwaksmanii, Alternaria alternata, Cladosporium herbarum, Phoma violacea,Stemphylium dentriticum, Candida albicans, Escherichia coli, andGlioclasium roseum. In another exemplary embodiment, there is a provisothat when the compound is present in a fungus, the fungus is not amember selected from Saccharomyces cerevisiae, Aspergillus niger,Fusarium solani, Penicillium pinophilum, Scopulariopsis brevicaulis,Streptoverticillium waksmanii, Alternaria alternata, Cladosporiumherbarum, Phoma violacea, Stemphylium dentriticum, Candida albicans, andGlioclasium roseum.

In an exemplary embodiment, the compound is present in a microorganismwhich is a member selected from a dermatophyte, Trichophyton,Microsporum, Epidermophyton and yeast-like fungi. In an exemplaryembodiment, there is a proviso that when the compound is present in ayeast-like fungus, that yeast-like fungus is not a member selected fromAspergillus niger and Candida albicans. In another exemplary embodiment,the microorganism is a member selected from a dermatophyte,Trichophyton, Microsporum, Epidermophyton and yeast-like fungi. In anexemplary embodiment, the microorganism is a dermatophyte. In anotherexemplary embodiment, the microorganism is a member selected fromTrichophyton species. In an exemplary embodiment, the microorganism is amember selected from is a member selected from T. rubrum and T.menagrophytes. In an exemplary embodiment, the microorganism is adermatophyte and said dermatophyte is a member selected from T. rubrumand T. menagrophytes.

In another exemplary embodiment, the compound is present in a human oran animal. In another exemplary embodiment, the compound is present in amicroorganism which is in, or on the surface of, a human or an animal.In another exemplary embodiment, the compound is present in amicrooganism which is present in a human nail unit of a human or a nail,hoof, or horn component of an animal. In another exemplary embodiment,the compound is present in a microoganism which is present in a memberselected from a human nail plate, human nail bed, proximal nail fold,lateral nail fold and combinations thereof. In another exemplaryembodiment, the compound is present in a microoganism which is presentin a member selected from a human nail plate and a human nail bed. Inanother exemplary embodiment, the compound is present in a microoganismwhich is present in a member selected from a proximal nail fold and alateral nail fold. In another exemplary embodiment, the microorganism isa member selected from dermatophyte, Trichophyton, Microsporum,Epidermophyton and yeast-like fungi. In another exemplary embodiment,wherein said compound is a dermatophyte. In another exemplaryembodiment, the dermatophyte is a member selected from T. rubrum and T.menagrophytes.

I. f) Formulations with Keratin

When a compound of the invention described herein is applied to a nailcomponent of a human, the compound absorbs or penetrates into the nail.The human nail is primarily composed of keratin (i.e. hair keratin orα-keratin) as well as trace amounts of lipid components. Therefore, inthe process of treating a disease of the nail or killing or inhibitingthe growth of a microorganism, a formulation comprising a human nailunit and a compound of the invention is formed.

In another aspect, the invention provides a formulation comprising: (a)a compound which is a member selected from a boron-containing compound,a 2′-amino ribofuranose-containing compound, a 3′-aminoribofuranose-containing compound, and combinations thereof; and (b) akeratin containing component which is a member selected from a humannail unit, skin and hair. In an exemplary embodiment, the compound ofpart (a) contacts the component of part (b). In an exemplary embodiment,the keratin containing component is a nail plate of the human nail unit.In an exemplary embodiment, the keratin containing component is a nailbed of the human nail unit. In an exemplary embodiment, the keratincontaining component is a proximal nail fold of the human nail unit. Inan exemplary embodiment, the keratin containing component is a lateralnail fold of the human nail unit. In another exemplary embodiment, thehuman nail unit comprises a member selected from keratin and lipid. Inanother exemplary embodiment, keratin is a member selected from skinkeratin and nail/hair keratin. In another exemplary embodiment, lipid isa member selected from cholesterol sulfate, cerebroside, ceramide, freesterol, free fatty acids, triglycerides, sterol esters, wax esters, andsqualene.

In an exemplary embodiment, the compound is present in the formulationat a concentration which is a member selected from about 0.001%, about0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about2%, about 2.5%, about 3%. In another exemplary embodiment, the keratinis present in said formulation at a concentration which is a memberselected from about 99.99%, about 99.95%, about 99.90%, about 99.5%,about 99.0%, about 98.5%, about 98.0%, about 97.5% and about 97%. Inanother exemplary embodiment, the compound is a compound describedherein. In another exemplary embodiment, the compound is as described inFormulae (I), (Ia) (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij),(Ik), (Il), (Im), (In), (Io), (Ip), (Iq), (Ir), (Is), (It), (Iu), (Iv),(Iw), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae), (Iaf), (Iag), (Iah),(Iai), (Iaj), (Iak), (II), (IIa), (IIb), (IIc), (IId), and (III). Inanother exemplary embodiment, the compound is an acyclic boronic esteras described herein. In another exemplary embodiment, the compound is amember selected from C1-C96 described herein. In another exemplaryembodiment, the compound is a member selected from a compound appearingin FIG. 19. In another exemplary embodiment, the compound is a memberselected from a compound appearing in FIG. 20. In another exemplaryembodiment, the compound is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In another exemplaryembodiment, 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole is presentis said formulation at a concentration which is a member selected fromabout 0.001%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about1%, and about 1.5%.

In another aspect, the invention provides a method of forming thisformulation, wherein said method comprises applying said compound to aformulation comprising keratin, thereby forming said formulation. In anexemplary embodiment, the formulation comprising keratin is a human nailunit. In an exemplary embodiment, the formulation comprising keratin isa member selected from a nail plate, nail bed, proximal nail fold, andlateral nail fold. Methods of making these formulations are described inthe Examples section.

I. g.) Preparation of Boron-Containing Editing Domain Inhibitors

Compounds of use in the present invention can be prepared usingcommercially available starting materials, known intermediates, or byusing the synthetic methods published in references described andincorporated by reference herein.

I. h.) Boronic Esters

The following exemplary schemes illustrate methods of preparingboron-containing molecules of the present invention. These methods arenot limited to producing the compounds shown, but can be used to preparea variety of molecules such as the compounds and complexes describedherein. The compounds of the present invention can also be synthesizedby methods not explicitly illustrated in the schemes but are well withinthe skill of one in the art. The compounds can be prepared using readilyavailable materials of known intermediates.

In the following schemes, the symbol X represents bromo or iodo. Thesymbol Y is selected from H, lower alkyl, and arylalkyl. The symbol Z isselected from H, alkyl, and aryl. The symbol PG represents protectinggroup. The symbols A, D, E, G, R^(x), R^(y), R^(z), R^(1a), R_(2a),R_(3a), R_(4a), R_(5a), R_(6a), R_(7a), R_(8a), R_(9a), R_(10a),R^(11a), and R^(12a) can be used to refer to the corresponding symbolsin the compounds described herein.

Boronic Acid Preparation Strategy #1

In Scheme 1, Step 1 and 2, compounds 1 or 2 are converted into alcohol3. In step 1, compound 1 is treated with a reducing agent in anappropriate solvent. Suitable reducing agents include borane complexes,such as borane-tetrahydrofuran, borane-dimethylsulfide, combinationsthereof and the like. Lithium aluminum hydride, or sodium borohydridecan also be used as reducing agents. The reducing agents can be used inquantities ranging from 0.5 to 5 equivalents, relative to compound 1 or2. Suitable solvents include diethyl ether, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane, combinations thereof and the like.Reaction temperatures range from 0° C. to the boiling point of thesolvent used; reaction completion times range from 1 to 24 h.

In Step 2, the carbonyl group of compound 2 is treated with a reducingagent in an appropriate solvent. Suitable reducing agents include boranecomplexes, such as borane-tetrahydrofuran, borane-dimethylsulfide,combinations thereof and the like. Lithium aluminum hydride, or sodiumborohydride can also be used as reducing agents. The reducing agents canbe used in quantities ranging from 0.5 to 5 equivalents, relative tocompound 2. Suitable solvents include lower alcohol, such as methanol,ethanol, and propanol, diethyl ether, tetrahydrofuran, 1,4-dioxane and1,2-dimethoxyethane, combinations thereof and the like. Reactiontemperatures range from 0° C. to the boiling point of the solvent used;reaction completion times range from 1 to 24 h.

In Step 3, the hydroxyl group of compound 3 is protected with aprotecting group which is stable under neutral or basic conditions. Theprotecting group is typically selected from methoxymethyl, ethoxyethyl,tetrahydropyran-2-yl, trimethylsilyl, tert-butyldimethylsilyl,tributylsilyl, combinations thereof and the like. In the case ofmethoxymethyl, compound 3 is treated with 1 to 3 equivalents ofchloromethyl methyl ether in the presence of a base. Suitable basesinclude sodium hydride, potassium tert-butoxide, tertiary amines, suchas diisopropylethylamine, triethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, and inorganic bases, such as sodiumhydroxide, sodium carbonate, potassium hydroxide, potassium carbonate,combinations thereof and the like. The bases can be used in quantitiesranging from 1 to 3 equivalents, relative to compound 3. Reactiontemperatures range from 0° C. to the boiling point of the solvent used;preferably between 0 and 40° C.; reaction completion times range from 1h to 5 days.

In the case of tetrahydropyran-2-yl, compound 3 is treated with 1 to 3equivalents of 3,4-dihydro-2H-pyran in the presence of 1 to 10 mol % ofacid catalyst. Suitable acid catalysts include pyridiniump-toluenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid,methanesulfonic acid, hydrogen chloride, sulfuric acid, combinationsthereof and the like. Suitable solvents include dichloromethane,chloroform, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, toluene,benzene, and acetonitrile combinations thereof and the like. Reactiontemperatures range from 0° C. to the boiling point of the solvent used;preferably between 0 and 60° C., and is complete in 1 h to 5 days.

In the case of trialkylsilyl, compound 3 is treated with 1 to 3equivalents of chlorotrialkylsilyane in the presence of 1 to 3equivalents of base. Suitable bases include tertiary amines, such asimidazole, diisopropylethylamine, triethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, combinations thereof and the like.Reaction temperatures range from 0° C. to the boiling point of thesolvent used; preferably between 0 and 40° C.; reaction completion timesrange from 1 to 48 h.

In Step 4, compound 4 is converted into boronic acid (5) through halogenmetal exchange reaction. Compound 4 is treated with 1 to 3 equivalentsof alkylmetal reagent relative to compound 4, such as n-butyllithium,sec-butyllithium, tert-butyllithium, isopropylmagnesium chloride or Mgturnings with or without an initiator such as diisobutylaluminum hydride(DiBAl), followed by the addition of 1 to 3 equivalents of trialkylborate relative to compound 4, such as trimethyl borate, triisopropylborate, or tributyl borate. Suitable solvents include tetrahydrofuran,ether, 1,4-dioxane, 1,2-dimethoxyethane, toluene, hexanes, combinationsthereof and the like. Alkylmetal reagent may also be added in thepresence of trialkyl borate. The addition of butyllithium is carried outat between −100 and 0° C., preferably at between −80 and −40° C. Theaddition of isopropylmagnesium chloride is carried out at between −80and 40° C., preferably at between −20 and 30° C. The addition of Mgturnings, with or without the addition of DiBAl, is carried out atbetween −80 and 40° C., preferably at between −35 and 30° C. Theaddition of the trialkyl borate is carried out at between −100 and 20°C. After the addition of trialkyl borate, the reaction is allowed towarm to room temperature, which is typically between −30 and 30° C. Whenalkylmetal reagent is added in the presence of trialkyl borate, thereaction mixture is allowed to warm to room temperature after theaddition. Reaction completion times range from 1 to 12 h. Compound 5 maynot be isolated and may be used for the next step without purificationor in one pot.

In Step 5, the protecting group of compound 5 is removed under acidicconditions to give compound of the invention. Suitable acids includeacetic acid, trifluoroacetic acid, hydrochloric acid, hydrobromic acid,sulfuric acid, p-toluenesulfonic acid and the like. The acids can beused in quantities ranging from 0.1 to 20 equivalents, relative tocompound 5. When the protecting group is trialkylsilyl, basic reagents,such as tetrabutylammonium fluoride, can also be used. Suitable solventsinclude tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, methanol,ethanol, propanol, acetonitrile, acetone, combination thereof and thelike. Reaction temperatures range from 0° C. to the boiling point of thesolvent used; preferably between 10° C. and reflux temperature of thesolvent; reaction completion times range from 0.5 to 48 h. The productcan be purified by methods known to those of skill in the art.

In another aspect, the invention provides a method of making atetrahydropyran-containing boronic ester, said ester having a structureaccording to the following formula:

wherein R¹ and R₂ are members independently selected from H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. R¹ and R², together with the atoms to whichthey are attached, can be optionally joined to form a 4- to 7-memberedring. R^(9a), R^(10a), R^(11a) and R^(12a) are members independentlyselected from H, OR*, NR*R**, SR*, —S(O)R*, —S(O)₂R*, —S(O)₂NR*R**,nitro, halogen, cyano, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. R* andR** is a member selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl. Themethod comprises: a) subjecting a first compound to Grignard ororganolithium conditions, said first compound having a structureaccording to the following formula:

-   -   b) contacting the product of step a) with a borate ester,        thereby forming said tetrahydropyran-containing boronic ester.        In an exemplary embodiment, halogen is a member selected from        iodo and bromo. In another exemplary embodiment, the borate        ester is a member selected from B(OR¹)₂(OR²), wherein R¹ and R²        are each members independently selected from H, substituted or        unsubstituted methyl, substituted or unsubstituted ethyl,        substituted or unsubstituted propyl, substituted or        unsubstituted isopropyl, substituted or unsubstituted butyl,        substituted or unsubstituted t-butyl, substituted or        unsubstituted phenyl and substituted or unsubstituted benzyl. R¹        and R², together with the atoms to which they are joined, can        optionally form a member selected from substituted or        unsubstituted dioxaborolane, substituted or unsubstituted        dioxaborinane and substituted or unsubstituted dioxaborepane. In        another exemplary embodiment, the borate ester is a member        selected from B(OR¹)₂(OR²), wherein R¹ and R², together with the        atoms to which they are joined, form a member selected from        dioxaborolane, substituted or unsubstituted        tetramethyldioxaborolane, substituted or unsubstituted        phenyldioxaborolane, dioxaborinane, dimethyldioxaborinane and        dioxaborepane. In another exemplary embodiment, the Grignard or        organolithium conditions further comprise diisobutyl aluminum        hydride. In another exemplary embodiment, the temperature of the        Grignard reaction does not exceed about 35° C. In another        exemplary embodiment, the temperature of the Grignard reaction        does not exceed about 40° C. In another exemplary embodiment,        the temperature of the Grignard reaction does not exceed about        45° C. In an exemplary embodiment, step (b) is performed at a        temperature of from about −30° C. to about −20° C. In another        exemplary embodiment, step (b) is performed at a temperature of        from about −35° C. to about −25° C. In another exemplary        embodiment, step (b) is performed at a temperature of from about        −50° C. to about −0° C. In another exemplary embodiment,        step (b) is performed at a temperature of from about −40° C. to        about −20° C. In another exemplary embodiment, the        tetrahydropyran-containing boronic ester is

In another aspect, the invention provides a method of making a compoundhaving a structure according to the following formula

said method comprising: a) subjecting a first compound to Grignard ororganolithium conditions, said first compound having a structureaccording to the following formula:

b) quenching said subjecting reaction with water and a organic acid,thereby forming said compound. In an exemplary embodiment, wherein saidorganic acid is a member selected from acetic acid. In another exemplaryembodiment, the quenching step is essentially not contacted with astrong acid. In another exemplary embodiment, the compound is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In another exemplaryembodiment, the compound is purified by recrystallization from arecrystallization solvent, wherein said recrystallization solventessentially does not contain acetonitrile. In an exemplary embodiment,the recrystallization solvent contains less than 2% acetonitrile. In anexemplary embodiment, the recrystallization solvent contains less than1% acetonitrile. In an exemplary embodiment, the recrystallizationsolvent contains less than 0.5% acetonitrile. In an exemplaryembodiment, the recrystallization solvent contains less than 0.1%acetonitrile. In an exemplary embodiment, the recrystallization solventcontains toluene and a hydrocarbon solvent. In an exemplary embodiment,the recrystallization solvent contains about 1:1 toluene:hydrocarbonsolvent. In an exemplary embodiment, the recrystallization solventcontains about 2:1 toluene:hydrocarbon solvent. In an exemplaryembodiment, the recrystallization solvent contains about 3:1toluene:hydrocarbon solvent. In an exemplary embodiment, therecrystallization solvent contains about 4:1 toluene:hydrocarbonsolvent. In an exemplary embodiment, the hydrocarbon solvent is a memberselected from heptane, octane, hexane, pentane and nonane. In anexemplary embodiment, the recrystallization solvent is 3:1toluene:heptane.

Boronic Acid Preparation Strategy #2

In Scheme 2, Step 6, compound 2 is converted into boronic acid (6) via atransition metal catalyzed cross-coupling reaction. Compound 2 istreated with 1 to 3 equivalents of bis(pinacolato)diboron or4,4,5,5-tetramethyl-1,3,2-dioxaborolane in the presence of transitionmetal catalyst, with the use of appropriate ligand and base asnecessary. Suitable transition metal catalysts include palladium(II)acetate, palladium(II) acetoacetonate,tetrakis(triphenylphosphine)palladium,dichlorobis(triphenylphosphine)palladium,[1,1′-bis(diphenylphosphino)ferrocen]dichloropalladium(II), combinationsthereof and the like. The catalyst can be used in quantities rangingfrom 1 to 5 mol % relative to compound 2. Suitable ligands includetriphenylphosphine, tri(o-tolyl)phosphine, tricyclohexylphosphine,combinations thereof and the like. The ligand can be used in quantitiesranging from 1 to 5 equivalents relative to compound 2. Suitable basesinclude sodium carbonate, potassium carbonate, potassium phenoxide,triethylamine, combinations thereof and the like. The base can be usedin quantities ranging from 1 to 5 equivalents relative to compound 2.Suitable solvents include N,N-dimethylformamide, dimethylsufoxide,tetrahydrofuran, 1,4-dioxane, toluene, combinations thereof and thelike. Reaction temperatures range from 20° C. to the boiling point ofthe solvent used; preferably between 50 and 150° C.; reaction completiontimes range from 1 to 72 h.

Pinacol ester is then oxidatively cleaved to give compound 6. Pinacolester is treated with sodium periodate followed by acid. Sodiumperiodate can be used in quantities ranging from 2 to 5 equivalentsrelative to compound 6. Suitable solvents include tetrahydrofuran,1,4-dioxane, acetonitrile, methanol, ethanol, combinations thereof andthe like. Suitable acids include hydrochloric acid, hydrobromic acid,sulfuric acid combinations thereof and the like. Reaction temperaturesrange from 0° C. to the boiling point of the solvent used; preferablybetween 0 and 50° C.; reaction completion times range from 1 to 72 h.

In Step 7, the carbonyl group of compound 6 is treated with a reducingagent in an appropriate solvent to give a compound of the invention.Suitable reducing agents include borane complexes, such asborane-tetrahydrofuran, borane-dimethylsulfide, combinations thereof andthe like. Lithium aluminum hydride, or sodium borohydride can also beused as reducing agents. The reducing agents can be used in quantitiesranging from 0.5 to 5 equivalents, relative to compound 6. Suitablesolvents include lower alcohol, such as methanol, ethanol, and propanol,diethyl ether, tetrahydrofuran, 1,4-dioxane and 1,2-dimethoxyethane,combinations thereof and the like. Reaction temperatures range from 0°C. to the boiling point of the solvent used; reaction completion timesrange from 1 to 24 h.

Boronic Acid Preparation Strategy #3

In Scheme 3, Step 8, compounds of the invention can be prepared in onestep from compound 3. Compound 3 is mixed with trialkyl borate thentreated with alkylmetal reagent. Suitable alkylmetal reagents includen-butyllithium, sec-butyllithium, tert-butyllithium combinations thereofand the like. Suitable trialkyl borates include trimethyl borate,triisopropyl borate, tributyl borate, combinations thereof and the like.The addition of butyllithium is carried out at between −100 and 0° C.,preferably at between −80 and −40° C. The reaction mixture is allowed towarm to room temperature after the addition. Reaction completion timesrange from 1 to 12 h. The trialkyl borate can be used in quantitiesranging from 1 to 5 equivalents relative to compound 3. The alkylmetalreagent can be used in quantities ranging from 1 to 2 equivalentsrelative to compound 3. Suitable solvents include tetrahydrofuran,ether, 1,4-dioxane, 1,2-dimethoxyethane, toluene, hexanes, combinationsthereof and the like. Reaction completion times range from 1 to 12 h.Alternatively, a mixture of compound 3 and trialkyl borate can berefluxed for 1 to 3 h and the alcohol molecule formed upon the esterexchange can be distilled out before the addition of alkylmetal reagent.

Boronic Acid Preparation Strategy #4

In Scheme 4, Step 10, the methyl group of compound 7 is brominated usingN-bromosuccinimide. N-bromosuccinimide can be used in quantities rangingfrom 0.9 to 1.2 equivalents relative to compound 7. Suitable solventsinclude carbon tetrachloride, tetrahydrofuran, 1,4-dioxane,chlorobenzene, combinations thereof and the like. Reaction temperaturesrange from 20° C. to the boiling point of the solvent used; preferablybetween 50 and 150° C.; reaction completion times range from 1 to 12 h.

In Step 11, the bromomethylene group of compound 8 is converted to thebenzyl alcohol 3. Compound 8 is treated with sodium acetate or potassiumacetate. These acetates can be used in quantities ranging from 1 to 10equivalents relative to compound 8. Suitable solvents includetetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide,combinations thereof and the like. Reaction temperatures range from 20°C. to the boiling point of the solvent used; preferably between 50 and100° C.; reaction completion times range from 1 to 12 h. The resultingacetate is hydrolyzed to compound 3 under basic conditions. Suitablebases include sodium hydroxide, lithium hydroxide, potassium hydroxide,combinations thereof and the like. The base can be used in quantitiesranging from 1 to 5 equivalents relative to compound 8. Suitablesolvents include methanol, ethanol, tetrahydrofuran, water, combinationsthereof and the like. Reaction temperatures range from 20° C. to theboiling point of the solvent used; preferably between 50 and 100° C.;reaction completion times range from 1 to 12 h. Alternatively, compound8 can be directly converted into compound 3 under the similar conditionabove.

Steps 3 through 5 convert compound 3 into a compound of the invention.

Boronic Acid Preparation Strategy #5

In Scheme 5, Step 12, compound 2 is treated with (methoxymethyl)triphenylphosphonium chloride or (methoxymethyl)triphenylphosphoniumbromide in the presence of base followed by acid hydrolysis to givecompound 9. Suitable bases include sodium hydride, potassiumtert-butoxide, lithium diisopropylamide, butyllithium, lithiumhexamethyldisilazane, combinations thereof and the like. The(methoxymethyl)triphenylphosphonium salt can be used in quantitiesranging from 1 to 5 equivalents relative to compound 2. The base can beused in quantities ranging from 1 to 5 equivalents relative to compound2. Suitable solvents include tetrahydrofuran, 1,2-dimethoxyethane,1,4-dioxane, ether, toluene, hexane, N,N-dimethylformamide, combinationsthereof and the like. Reaction temperatures range from 0° C. to theboiling point of the solvent used; preferably between 0 and 30° C.;reaction completion times range from 1 to 12 h. The enolether formed ishydrolyzed under acidic conditions. Suitable acids include hydrochloricacid, hydrobromic acid, sulfuric acid, and the like. Suitable solventsinclude tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, methanol,ethanol, combination thereof and the like. Reaction temperatures rangefrom 20° C. to the boiling point of the solvent used; preferably between50 and 100° C.; reaction completion times range from 1 to 12 h.

Steps 2 through 5 convert compound 9 into a compound of the invention.

Boronic Acid Preparation Strategy #6

In Scheme 6, compound (I) wherein R¹ is H is converted into compound (I)wherein R¹ is alkyl by mixing with the corresponding alcohol, R¹OH. Thesuitable solvents include tetrahydrofuran, 1,2-dimethoxyethane,1,4-dioxane, toluene, combinations thereof and the like. The alcohol(R¹OH) can be used as the solvent as well. Reaction temperatures rangefrom 20° C. to the boiling point of the solvent used; preferably between50 and 100° C.; reaction completion times range from 1 to 12 h.

Boronic Acid Preparation Strategy #7

In Scheme 7, compound (Ia) is converted into its aminoalcohol complex(Ib). Compound (Ia) is treated with HOR¹NR^(1a)R^(1b). The aminoalcoholcan be used in quantities ranging from 1 to 10 equivalents relative tocompound (Ia). Suitable solvents include methanol, ethanol, propanol,tetrahydrofuran, acetone, acetonitrile, 1,2-dimethoxyethane,1,4-dioxane, toluene, N,N-dimethylformamide, water, combination thereofand the like. Reaction temperatures range from 20° C. to the boilingpoint of the solvent used; preferably between 50 and 100° C.; reactioncompletion times range from 1 to 24 h.

The compounds of the invention can be converted into hydrates andsolvates by methods similar to those described above.

I. h.) Borinic Esters

Methods of making borinic esters are known in the art, and it is withinthe knowledge of one skilled in the art to use these methods in order tomake the boronic esters described herein. Examples include U.S. patentSer. No. 10/868,268 and U.S. Prov. Pat. No. ______ (Attorney Docket No.64507-5021PR, filed May 2, 2006) which are herein incorporated byreference.

I. i.) 2′-Amino or 3′-Amino Ribofuranoses

Methods of making 2′-amino ribofuranoses or 3′-amino ribofuranoses areknown in the art, and it is within the knowledge of one skilled in theart to use these methods in order to make the 2′-amino ribofuranosesdescribed herein.

Ashton et al. (Can. Pat. App. 2,031,644 (1991)) and Durette, et al. (UKPat. App. 2,207,678 (1989)) disclose the synthesis of the amino acidstarting material for compound D5. Hardee, et al., (PCT Int. App.WO2005020885 (2005)) discloses the synthesis of the nucleoside startingmaterial for compound D6. Sakthivel, (Sakthivel, et al., Tet. Let.46(22): 3883-3887 (2005)) Sartorelli, et al., (U.S. Pat. Appl. Pub.2004116362); Roberts, et al., (PCT Int. Appl. WO2003093290); Liu, etal., Nucleosides, Nucleotides & Nucleic Acids, 20(12): 1975-2000 (2001);Minakawa, et al., J. Org. Chem., 64(19): 7158-7172 (1999); Daelemans, etal., Molecular Pharmacology, 52(6): 1157-1163 (1997) all disclose thesynthesis of the nucleoside starting material for compound D7.

Examples of how to prepare these compounds is shown below:

Compounds 1-14 are produced by a final step (Lincecum, T. L. et al., S.Molecular Cell, 11: 951-963 (2003); Kim, B.-T. et al., J. Bull. KoreanChem. Soc., 25: 243-248 (2004)):

Compounds 15-18 are produced by a final step (See Lincecum):

Methods for preparing dimers, trimers and higher homologs of smallorganic molecules, such as those of use in the present invention, aswell as methods of functionalizing a polyfunctional framework moleculeare well known to those of skill in the art. For example, an aromaticamine of the invention is converted to the corresponding isothiocyanateby the action of thiophosgene. The resulting isothiocyanate is coupledto an amine of the invention, thereby forming either a homo- orheterodimeric species. Alternatively, the isothiocyanate is coupled withan amine-containing backbone, such as polylysine, thereby forming aconjugate between a polyvalent framework and a compound of theinvention. If it is desired to prepare a heterofunctionalized polyvalentspecies, the polylysine is underlabeled with the first isothiocyanateand subsequently labeled with one or more different isothiocyanates.Alternatively, a mixture of isothiocyanates is added to the backbone.Purification proceeds by, for example, size exclusion chromatography,dialysis, nanofiltration and the like.

II. Assays for Inhibitors of tRNA Synthetase Editing Domains

Art-recognized techniques of genetics and molecular biology are of useto identify compounds that bind to and/or inhibit the editing domain ofa tRNA synthetase. Moreover, these techniques are of use to distinguishwhether a compound binds to and/or inhibits the synthetic domain, theediting domain, or both the editing and synthetic domains.

In an exemplary assay, activity of a representative compound against theediting domain was confirmed. To identify the target of the novelboron-containing antifungal compound C10, mutants in S. cerevisiaeshowing resistance to compound C10 were isolated. Characterization of 11mutants showed that they have an 8-64 fold increase in resistance to C10over wildtype. The mutants were furthermore shown to be sensitive tovarious antifungal agents with known modes of action, suggesting thatthe cellular target of C10 is distinct from the target of the otherantifungal agents. Isolation of three different plasmids bearing CDC60from plasmid libraries generated from three independently isolatedmutants implicated CDC60, the gene for the cytoplasmic leucyl-tRNAsynthetase in resistance against C10. Sequence analysis of CDC60 fromthe 11 mutants revealed that the mutations were all located in theediting domain of this enzyme. In a further series of experiments,additional copies of the CDC60 gene were introduced in S. cerevisiae,which gave rise to an eight-fold increase in resistance to C10. Thesefindings confirm a strong link between the editing activity of theenzyme and the inhibition of C10, which entails a novel mechanism oftRNA synthetase inhibition.

Assays to determine whether, and how effectively, a particular compoundbinds to and/or inhibits the editing domain of a selected tRNAsynthetase are also set forth herein, and additional assays are readilyavailable to those of skill in the art. Briefly, in an exemplary assay,an improperly charged tRNA and a tRNA synthetase that is capable ofediting the improperly charged tRNA are combined. The resulting mixtureis contacted with the putative inhibitor and the degree of editinginhibition is observed.

Another assay uses genetics to show that a drug works via the editingdomain. In this assay, the compound is first tested against a strain ofcells over-expressing copies of the tRNA synthetase gene. The compound'seffect on the over-expressing strain is compared with a control strainto determine whether the compound is active against the synthetase. Ifthe minimum inhibitory concentration (MIC) is 2-fold higher in thestrain with extra copies of the synthetase gene than the MIC of theinhibitor against a wild type cell, a further genetic screen isconducted to determine whether the increased resistance is due tomutations in the editing domain. In this second screen, the controlstrain is challenged against a high concentration of the inhibitor. Thecolonies surviving the challenge are isolated and DNA from these cellsis isolated. The editing domain is amplified using a proof-reading PCRenzyme and the appropriate primers. The PCR product can be purifiedusing standard procedures. The sequence amplified mutant DNA is comparedto wild-type. If the mutant DNA bears mutations in the editing domain,such results would suggest that the compound binds to the editing domainand affects the editing function of the molecule through this domain.

The assays set forth above are useful in essentially any microbialsystem, e.g., bacterial, fungal, parasitic, viral and the like.

Generally, the compounds to be tested are present in the assays inranges from about 1 pM to about 100 mM, preferably from about 1 pM toabout 1 μM. Other compounds range from about 1 nM to about 100 nM,preferably from about 1 nM to about 1 μM.

The effects of the test compounds upon the function of the enzymes canalso be measured by any suitable physiological change. When thefunctional consequences are determined using intact cells or animals,one can also measure a variety of effects such as transmitter release,hormone release, transcriptional changes to both known anduncharacterized genetic markers, changes in cell metabolism such as cellgrowth or pH changes, and changes in intracellular second messengerssuch as Ca²⁺, or cyclic nucleotides.

High throughput screening (HTS) is also of use in identifying promisingcandidates of the invention.

Utilizing the assays set forth herein and others readily available inthe art, those of skill in the art will be able to readily and routinelydetermine other compounds and classes of compounds that operate to bindto and/or inhibit the editing domain of tRNA synthetases.

In another aspect, the invention provides a method for identifying acompound which binds to an editing domain of a tRNA synthetasecomprising: a) contacting said editing domain with a test compound underconditions suitable for binding; and b) detecting binding of said testcompound to said editing domain. In an exemplary embodiment, detectingbinding of said compound comprises use of at least one detectableelement, isotope, or chemical label attached to said compound. In anexemplary embodiment, the element, isotope or chemical label is detectedby a fluorescent, luminescent, radioactive, or absorbance readout. In anexemplary embodiment, the contacting of said test compound with saidediting domain also includes further contacting said test compound andsaid editing domain with a member selected from AMP and a molecule witha terminal adenosine. In an exemplary embodiment, said tRNA synthetaseis derived from a member selected from alanyl tRNA synthetase, isoleucyltRNA synthetase, leucyl tRNA synthetase, methionyl tRNA synthetase,lysyl tRNA synthetase, phenylalanyl tRNA synthetase, prolyl tRNAsynthetase, threonyl tRNA synthetase and valyl tRNA synthetase. In anexemplary embodiment, the tRNA synthetase is derived from leucyl tRNAsynthetase. In an exemplary embodiment, the tRNA synthetase is derivedfrom a mutated tRNA synthetase, wherein said mutated tRNA synthetasecomprises amino acid mutations in an editing domain. In anotherexemplary embodiment, the mutated tRNA synthetase comprises amino acidmutations in the editing domain as listed in Table 4. In anotherexemplary embodiment, wherein said editing domain of a tRNA synthetasecomprises the amino acid sequence of SEQ ID NOS: 1-15.

In another aspect, the invention provides a method for identifying acompound which binds to an editing domain of a tRNA synthetase, saidassay comprising: a) contacting said editing domain of a tRNA synthetasewith said compound under conditions suitable for binding of saidcompound with said editing domain of a tRNA synthetase; b) comparing abiological activity of said editing domain of a tRNA synthetasecontacting said compound to said biological activity when not contactingsaid compound; and c) identifying said compound as binding to saidediting domain of a tRNA synthetase if said biological activity of saidediting domain of a tRNA synthetase is reduced when contacting saidcompound. In an exemplary embodiment, the biological activity ishydrolysis of noncognate amino acid. In another exemplary embodiment,the hydrolysis of said noncognate amino acid is detected through the useof one or more labels. In another exemplary embodiment, the labelsinclude a radiolabel, a fluorescent marker, an antibody, or acombination thereof. In another exemplary embodiment, said labels can bedetected using spectroscopy. In another exemplary embodiment, theediting domain of a tRNA synthetase is derived from a member selectedfrom alanyl tRNA synthetase, isoleucyl tRNA synthetase, leucyl tRNAsynthetase, methionyl tRNA synthetase, lysyl tRNA synthetase,phenylalanyl tRNA synthetase, prolyl tRNA synthetase, threonyl tRNAsynthetase and valyl tRNA synthetase. In another exemplary embodiment,said editing domain of a tRNA synthetase is derived from leucyl tRNAsynthetase.

In another aspect, the invention provides a method of generating tRNAmolecules with noncognate amino acid comprising: a) creating orisolating a mutated tRNA synthetase with altered amino acid editingdomains; and b) contacting a tRNA molecule with said mutated tRNAsynthetase and a noncognate amino acid. In another exemplary embodiment,the mutated tRNA synthetase contains one or more amino acid mutations inan editing domain. In another exemplary embodiment, the mutated tRNAsynthetase is unable to bind with1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In another exemplaryembodiment, the mutated tRNA synthetase is able to bind with1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole.

In another aspect, the invention provides a composition that comprisesone or more tRNA molecules attached to noncognate amino acids, whereinsaid tRNA molecules are synthesized using one or more mutated tRNAsynthetases isolated from a microorganism or a cell line derived from amicroorganism. In an exemplary embodiment, the microorganism is a fungusor a yeast. In an exemplary embodiment, wherein said mutated tRNAsynthetases contain amino acid mutations in their editing domains. In anexemplary embodiment, said mutated tRNA synthetases comprise pointmutations in the editing domain as listed in Table 4.

III. Amino Acid and Nucleotide Sequences Used in Assays

tRNA Sequences that Interact with the tRNA Synthetase-C10-AMP Complex

Transfer RNAs (tRNAs) translate mRNA into a protein on a ribosome. Eachtransfer RNA contains an anti-codon region that hybridizes with mRNA,and an amino acid which may be attached to the growing peptide. Thestructural gene of tRNA is about 72 to 90 nucleotides long and foldsinto a cloverleaf structure (Sharp S. J., Schaack J., Coolen L., BurkeD. J. and Soll D., “Structure and transcription of eukaryotic tRNAgenes”, Crit. Rev. Biochem, 19:107 144 (1985); Geiduschek E. O., andTocchini-Valentini, “Transcription by RNA polymerase III”, Annu. Rev.Biochem. 57:873 914 (1988)).

In one embodiment, C10 contacts AMP and a tRNA synthetase, and the tRNAsynthetase in turn contacts a tRNA molecule. In another embodiment, C10contacts AMP from the tRNA molecules and a tRNA synthetase. Thenucleotide sequence of the tRNA molecule can be determined by theidentity of the tRNA synthetase involved. For example, for leucyl tRNAsynthetase, the cognate tRNA molecule bound will be tRNA-leucine (SEQ IDNO: 3), but a noncognate tRNA, such as isoleucine, (SEQ ID NO: 4) may bebound under certain conditions. In this and other embodiments, the term“noncognate” is meant to encompass both the singular and plural forms ofthe word, i.e. the phrase “noncognate amino acid” comprises one or moreamino acids.

SEQ ID NO: 3 corresponds to the nucleotide sequence of the tRNA-Leu genefrom Saccharomyces cerevisiae: gggagtttgg ccgagtggtt taaggcgtcagatttaggct ctgatatctt cggatgcaagggttcgaatc ccttagctct cacca

SEQ ID NO: 4 corresponds to the nucleotide sequence of the tRNA-Ile genefrom Saccharomyces cerevisiae: gaaactataa ttcaattggt tagaatagtattttgataag gtacaaatat aggttcaatc cctgttagtt tcatcca

Polypeptides Used in Binding and Inhibition Assays

In some binding and inhibition assays, it is more effective to use aportion of a tRNA synthetase molecule rather than the whole proteinitself. In such assays, polypeptides derived from tRNA synthetases areused in the experiment.

In one preferred embodiment, polypeptide fragments corresponding to theediting domain of a tRNA synthetase molecule are used in assay andbinding experiments. Two such fragments are represented by SEQ ID NO: 1and SEQ ID NO:2.

SEQ ID NO 1: TPQEYIGVKIEALEFADDAAKIIDSSSDLDKSKKFYFVAATLRPETMYGQTCCFVSPTIEYGIFDAGDSYFITTERAFKNMSYQKLTPKRGFYKPIVTVPGKAFIGTKIHAPQSVYPELRILPMETVIATKGTGVVTCVPSNSPDDYITTKDLLHKPEYYGIKPEWIDHEIVPIMHTEKYGDLTAKAIVEEKKIQSPKDKNLLAEAKKIAYKEDYYTGTMIYGPYKGEKVEQAKNKVKADMIAAG EAFVYNEPESQDPSEQ ID NO 2: MTPQEYIGVKIEALEFADDAAKIIDSSSDLDKSKKFYFVAATLRPETMYGQTCCFVSPTIEYGIFDAGDSYFITTERAFKNMSYQKLTPKRGFYKPIVTVPGKAFIGTKIHAPQSVYPELRILPMETVIATKGTGVVTCVPSNSPDDYITTKDLLHKPEYYGIKPEWIDHEIVPIMHTEKYGDLTAKAIVEEKKIQSPKDKNLLAEAKKIAYKEDYYTGTMIYGPYKGEKVEQAKNKVKADMIAAGEAFVYNEPESQDPQDPNSSSVDKLAAALEHHHHHIV. Methods for Inhibiting the Editing Domain of tRNA Synthetase

According to another aspect of the invention, a method for binding toand/or inhibiting the editing domain of a tRNA synthetase is providedwhich comprises contacting a tRNA synthetase with a compound thatinhibits the editing domain under the conditions in which the tRNAsynthetase interacts with its substrate to form an aminoacyl adenylateintermediate and, preferably, to form a charged tRNA. Such conditionsare known to those skilled in the art. In an exemplary embodiment, thecompound is one described herein. The tRNA synthetase is contacted withan amount of inhibitor sufficient to result in a detectable amount oftRNA synthetase inhibition. This method can be performed on a tRNAsynthetase that is contained within an organism or which is outside anorganism. In an exemplary embodiment, the method is performed on a tRNAsynthetase that is contained within a microorganism or a microbial cellthat is in, or on the surface of, a human or an animal. The methodresults in a decrease in the amount of charged tRNA produced by the tRNAsynthetase that has an inhibited editing domain. In an exemplaryembodiment, the inhibition takes place in a cell, such as a microbialcell. In another exemplary embodiment, the microbial cell is a bacteria,fungus, yeast or parasite. In another exemplary embodiment, the tRNAsynthetase is a mitochondrial tRNA synthetase or a cytoplasmic tRNAsynthetase.

In an exemplary embodiment, the invention provides a method ofinhibiting conversion of a tRNA molecule into a charged tRNA molecule.The method involves contacting a tRNA synthetase with a compoundeffective to inhibit activity of an editing domain of said tRNAsynthetase, under conditions sufficient to inhibit said activity,thereby inhibiting said conversion wherein the compound is a memberselected from those compounds described herein. In an exemplaryembodiment, the compound is a member selected from a cyclic boronicester, cyclic borinic ester, 2′-amino ribofuranose moiety and a 3′-aminoribofuranose moiety. In an exemplary embodiment, the inhibition occurswithin a cell, and the cell is a microbial cell. In another exemplaryembodiment, the microbial cell is a member selected from a bacteria,fungus, yeast, and parasite. In an exemplary embodiment, the tRNAsynthetase is a member selected from a mitochondrial tRNA synthetase anda cytoplasmic tRNA synthetase. In another exemplary embodiment, the tRNAsynthetase is a member selected from alanyl tRNA synthetase, isoleucyltRNA synthetase, leucyl tRNA synthetase, methionyl tRNA synthetase,lysyl tRNA synthetase, phenylalanyl tRNA synthetase, prolyl tRNAsynthetase, threonyl tRNA synthetase and valyl tRNA synthetase. Inanother exemplary embodiment, the compound has a K_(D, synthesis) is ofgreater than 100 μM against a synthetic domain of said tRNA synthetase.

In certain embodiments, the mechanism of action of the compound is toinhibit the conversion of a tRNA molecule into a charged tRNA moleculeby binding to and/or inhibiting at least the editing domain of thesynthetase. The compounds of use in this method may also inhibit orotherwise interact with the synthetic domain (e.g., the active site ofthe synthetic domain). In a presently preferred embodiment, the editingdomain is inhibited selectively in the presence of the synthetic domain.In a preferred embodiment, the synthetic domain is essentiallyuninhibited, while the editing domain is inhibited at least 50%,preferably at least 60%, more preferably at least 70%, still morepreferably, at least 80% and even still more preferably at least 90% ofthe activity of the tRNA synthetase. In another preferred embodiment,the synthetic domain is inhibited by at most 50%, preferably at most30%, preferably at most 20%, 10%, preferably at most 8%, more preferablyat most 5%, still more preferably, at most 3% and even still morepreferably at most 1%. Inhibition of the editing domain produces adecrease in the amount of the properly charged tRNA which results inretardation or cessation of cell growth and division.

In another exemplary embodiment, the ratio of a minimum concentration ofsaid compound inhibiting said editing domain to a minimum concentrationof said compound inhibiting said synthetic domain of said tRNAsynthetase, represented as K_(D, edit)/K_(D, synthesis), is less thanone. In another exemplary embodiment, the K_(D, edit)/K_(D, synthesis)of the compound is a member selected from less than 0.5, less than 0.1and less than 0.05.

V. Methods of Inhibiting Microorganism Growth or Killing Microorganisms

In a further aspect, the invention provides a method for inhibiting thegrowth, or killing, a microorganism, preferably a bacteria, fungus,virus, yeast or parasite, comprising contacting the microorganism withan inhibitor of a tRNA synthetase, e.g., a compound described by aformula listed herein, under conditions which permit entry of thecompound into the organism. In a further aspect, the invention providesa method for inhibiting the growth, or killing, a microorganism,preferably a bacteria, fungus, virus, yeast or parasite, comprisingcontacting the microorganism with a compound which is a member selectedfrom Formulae (I), (Ia), (Ib), (Ic), (Id) (Ie), (If), (Ig), (Ih) (Ii),(Ij), (Ik), (Il) (Im), (In), (Io), (Ip) (Iq), (Ir), (Is), (It), (Iu),(Iv), (Iw), (Ix) (Iy), (Iz), (Iaa), (Iab), (Iac), (Iad), (Iae), (Iaf),(Iag), (Iah), (Iai), (Iaj), (Iak), (II), (IIa), (IIb), (IIc), (IId),(III), (VIII), (VIIIa), (VIIIb), (VIIIc), (VIIId), (VIIIe), (IX) e.g., acompound described by a formula listed herein, under conditions whichpermit entry of the compound into the organism. In a further aspect, theinvention provides a method for inhibiting the growth, or killing, amicroorganism, preferably a bacteria, fungus, virus, yeast or parasite,comprising contacting the microorganism with a compound which isdescribed in either FIG. 19 or FIG. 20 e.g., a compound described by aformula listed herein, under conditions which permit entry of thecompound into the organism. In an exemplary embodiment, the compoundinhibits the tRNA synthetase through the editing domain of thesynthetase. Such conditions are known to one skilled in the art andspecific conditions are set forth in the Examples appended hereto. Thismethod involves contacting a microbial cell with atherapeutically-effective amount of an editing domain inhibitor toinhibit tRNA synthetase in vivo or in vitro.

In another aspect, the invention provides a method of inhibiting thegrowth of a microorganism, or killing a microorganism, or both,comprising contacting the microorganism with a compound describedherein. Microorganisms are members selected from fungi, yeast, viruses,bacteria and parasites. In another exemplary embodiment, themicroorganism is inside, or on the surface of an animal. In an exemplaryembodiment, the animal is a member selected from human, cattle, deer,reindeer, goat, honey bee, pig, sheep, horse, cow, bull, dog, guineapig, gerbil, rabbit, cat, camel, yak, elephant, ostrich, otter, chicken,duck, goose, guinea fowl, pigeon, swan, and turkey. In another exemplaryembodiment, the animal is a human.

In an exemplary embodiment, the microorganism is a member selected froma fungus and a yeast. In another exemplary embodiment, the fungus oryeast is a member selected from Candida species, Trichophyton species,Microsporium species, Aspergillus species, Cryptococcus species,Blastomyces species, Cocciodiodes species, Histoplasma species,Paracoccidiodes species, Phycomycetes species, Malassezia species,Fusarium species, Epidermophyton species, Scytalidium species,Scopulariopsis species, Alternaria species, Penicillium species,Phialophora species, Rhizopus species, Scedosporium species andZygomycetes class. In another exemplary embodiment, the fungus or yeastis a member selected from Aspergilus fumigatus (A. fumigatus),Blastomyces dermatitidis, Candida Albicans (C. albicans, bothfluconazole sensitive and resistant strains), Candida glabrata (C.glabrata), Candida krusei (C. krusei), Cryptococcus neoformans (C.neoformans), Candida parapsilosis (C. parapsilosis), Candida tropicalis(C. tropicalis), Cocciodiodes immitis, Epidermophyton floccosum (E.floccosum), Fusarium solani (F. solani), Histoplasma capsulatum,Malassezia furfur (M. furfur), Malassezia pachydermatis (M.pachydermatis), Malassezia sympodialis (M. sympodialis), Microsporumaudouinii (M. audouinii), Microsporum canis (M. canis), Microsporumgypseum (M. gypseum), Paracoccidiodes brasiliensis and Phycomycetes spp,Trichophyton mentagrophytes (T. mentagrophytes), Trichophyton rubrum (T.rubrum), Trichophyton tonsurans (T. tonsurans). In another exemplaryembodiment, the fungus or yeast is a member selected from Trichophytonconcentricum, T. violaceum, T. schoenleinii, T. verrucosum, T.soudanense, Microsporum gypseum, M. equinum, Candida guilliermondii,Malassezia globosa, M. obtuse, M. restricta, M. slooffiae, andAspergillus flavus. In another exemplary embodiment, the fungus or yeastis a member selected from dermatophytes, Trichophyton, Microsporum,Epidermophyton and yeast-like fungi.

In an exemplary embodiment, the microorganism is a bacteria. In anexemplary embodiment, the bacteria is a gram-positive bacteria. Inanother exemplary embodiment, the gram-positive bacteria is a memberselected from Staphylococcus species, Streptococcus species, Bacillusspecies, Mycobacterium species, Corynebacterium species(Propionibacterium species), Clostridium species, Actinomyces species,Enterococcus species and Streptomyces species. In another exemplaryembodiment, the bacteria is a gram-negative bacteria. In anotherexemplary embodiment, the gram-negative bacteria is a member selectedfrom Acinetobacter species, Neisseria species, Pseudomonas species,Brucella species, Agrobacterium species, Bordetella species, Escherichiaspecies, Shigelia species, Yersinia species, Salmonella species,Klebsiella species, Enterobacter species, Haemophilus species,Pasteurella species, Streptobacillus species, spirochetal species,Campylobacter species, Vibrio species and Helicobacter species. Inanother exemplary embodiment, the bacterium is a member selected fromPropionibacterium acnes; Staphylococcus aureus; Staphylococcusepidermidis, Staphylococcus saprophyticus; Streptococcus pyogenes;Streptococcus agalactiae; Streptococcus pneumoniae; Enterococcusfaecalis; Enterococcus faecium; Bacillus anthracis; Mycobacteriumavium-intracellulare; Mycobacterium tuberculosis, Acinetobacterbaumanii; Corynebacterium diphtheria; Clostridium perfringens;Clostridium botulinum; Clostridium tetani; Clostridium difficile;Neisseria gonorrhoeae; Neisseria meningitidis; Pseudomonas aeruginosa;Legionella pneumophila; Escherichia coli; Yersinia pestis; Haemophilusinfluenzae; Helicobacter pylori; Campylobacter fetus; Campylobacterjejuni; Vibrio cholerae; Vibrio parahemolyticus; Trepomena pallidum;Actinomyces israelii; Rickettsia prowazekii; Rickettsia rickettsii;Chlamydia trachomatis; Chlamydia psittaci; Brucella abortus;Agrobacterium tumefaciens; and Francisella tularensis.

In an exemplary embodiment, the microorganism is a bacteria, which is amember selected from acid-fast bacterium, including Mycobacteriumspecies; bacilli, including Bacillus species, Corynebacterium species(also Propionibacterium) and Clostridium species; filamentous bacteria,including Actinomyces species and Streptomyces species; bacilli, such asPseudomonas species, Brucella species, Agrobacterium species, Bordetellaspecies, Escherichia species, Shigella species, Yersinia species,Salmonella species, Klebsiella species, Enterobacter species,Haemophilus species, Pasteurella species, and Streptobacillus species;spirochetal species, Campylobacter species, Vibrio species; andintracellular bacteria including Rickettsiae species and Chlamydiaspecies.

In an exemplary embodiment, the microorganism is a virus. In anexemplary embodiment, the virus is a member selected from hepatitis A-B,human rhinoviruses, Yellow fever virus, human respiratory coronaviruses,Severe acute respiratory syndrome (SARS), respiratory syncytial virus,influenza viruses, parainfluenza viruses 1-4, human immunodeficiencyvirus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Herpes simplexvirus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), human cytomegalovirus(HCMV), Varicella zoster virus, Epstein-Barr (EBV), polioviruses,coxsackieviruses, echoviruses, rubella virus, neuroderma-tropic virus,variola virus, papoviruses, rabies virus, dengue virus, West Nile virusand SARS virus. In another exemplary embodiment, the virus is a memberselected from picornaviridae, flaviviridae, coronaviridae,paramyxoviridae, orthomyxoviridae, retroviridae, herpesviridae andhepadnaviridae. In another exemplary embodiment, the virus is a memberselected from a virus included in the following table:

TABLE A Viruses Virus Category Pertinent Human Infections RNA VirusesPicomaviridae Polio Human hepatitis A Human rhinovirus Togaviridae andRubella—German measles Flaviviridae Yellow fever Coronaviridae Humanrespiratory coronavirus (HCV) Severe acute respiratory syndrome (SAR)Rhabdoviridae Lyssavirus—Rabies Paramyxoviridae Paramyxovirus—MumpsMorbillvirus—measles Pneumovirus—respiratory syncytial virusOrthomyxoviridae Influenza A-C Bunyaviridae Bunyavirus—Bunyamwera (BUN)Hantavirus—Hantaan (HTN) Nairevirus—Crimean-Congo hemorrhagic fever(CCHF) Phlebovirus—Sandfly fever (SFN) Uukuvirus—Uukuniemi (UUK) RiftValley Fever (RVFN) Arenaviridae Junin—Argentine hemorrhagic feverMachupo—Bolivian hemorrhagic fever Lassa—Lassa fever LCM—asepticlymphocyctic choriomeningitis Reoviridae Rotovirus Reovirus OrbivirusRetroviridae Human immunodeficiency virus 1 (HIV-1) Humanimmunodeficiency virus 2 (HIV-2) Simian immunodeficiency virus (SIV) DNAViruses Papovaviridae Pediatric viruses that reside in kidneyAdenoviridae Human respiratory distress and some deep-seated eyeinfections Parvoviridae Human gastro-intestinal distress (Norwalk Virus)Herpesviridae Herpes simplex virus 1 (HSV-1) Herpes simplex virus 2(HSV-2) Human cytomegalovirus (HCMV) Varicella zoster virus (VZV)Epstein-Barr virus (EBV) Human herpes virus 6 (HHV6) PoxviridaeOrthopoxvirus is sub-genus for smallpox Hepadnaviridae Hepatitis B virus(HBV) Hepatitis C virus (HCV)

In another exemplary embodiment, the microorganism is a parasite. In anexemplary embodiment, the parasite is a member selected from Plasmodiumfalciparum, P. vivax, P. ovale P. malariae, P. berghei, Leishmaniadonovani, L. infantum, L. chagasi, L. mexicana, L. amazonensis, L.venezuelensis, L. tropics, L. major, L. minor, L. aethiopica, L. Bianabraziliensis, L. (V.) guyanensis, L. (V.) panamensis, L. (V.) peruviana,Trypanosoma brucei rhodesiense, T. brucei gambiense, T. cruzi, Giardiaintestinalis, G. lambda, Toxoplasma gondii, Entamoeba histolytica,Trichomonas vaginalis, Pneumocystis carinii, and Cryptosporidium parvum.

VI. Methods of Treating or Preventing Infections

In another aspect, the invention provides a method of treating orpreventing an infection. The method includes administering to the animala therapeutically effective amount of the compound of the invention,sufficient to treat or prevent said infection. In an exemplaryembodiment, the compound is a compound described herein. In anotherexemplary embodiment, the compound has a structure according to Formulae(I) to (Iak) and (II) to (XI). In another exemplary embodiment, thecompound has a structure which is described in FIG. 19. In anotherexemplary embodiment, the compound has a structure which is described inFIG. 20. In another exemplary embodiment, the animal is a memberselected from human, cattle, deer, reindeer, goat, honey bee, pig,sheep, horse, cow, bull, dog, guinea pig, gerbil, rabbit, cat, camel,yak, elephant, ostrich, otter, chicken, duck, goose, guinea fowl,pigeon, swan, and turkey. In another exemplary embodiment, the animal isa human.

In another exemplary embodiment, the animal is a member selected from ahuman, cattle, goat, pig, sheep, horse, cow, bull, dog, guinea pig,gerbil, rabbit, cat, chicken and turkey. In another exemplaryembodiment, the infection is a member selected from a systemicinfection, a cutaneous infection, and an ungual, periungual or subungualinfection.

In another exemplary embodiment, the treatment of a disorder orcondition occurs through inhibition of an editing domain of an aminoacyltRNA synthetase.

VI. a) Methods of Treating of Preventing Ungual and/or PeriungualInfections

In another aspect, the invention provides a method of treating orpreventing an ungual and/or periungual infection. The method includesadministering to the animal a therapeutically effective amount of acompound or pharmaceutical formulation of the invention, sufficient totreat or prevent said infection. In another exemplary embodiment, themethod includes administering the compound or pharmaceutical formulationof the invention at a site which is a member selected from the skin,nail, hair, hoof, claw and the skin surrounding the nail, hair, hoof andclaw.

VI. a) 1) Onychomycosis

Onychomycosis is a disease of the nail caused by yeast, dermatophytes,or other molds, and represents approximately 50% of all nail disorders.Toenail infection accounts for approximately 80% of onychomycosisincidence, while fingernails are affected in about 20% of the cases.Dermatophytes are the most frequent cause of nail plate invasion,particularly in toenail onychomycosis. Onychomycosis caused by adermatophyte is termed Tinea unguium. Trichophyton rubrum is by far themost frequently isolated dermatophyte, followed by T. mentagrophytes.Distal subungual onychomycosis is the most common presentation of tineaunguium, with the main site of entry through the hyponychium (thethickened epidermis underneath the free distal end of a nail)progressing in time to involve the nail bed and the nail plate.Discoloration, onycholysis, and accumulation of subungual debris andnail plate dystrophy characterize the disease. The disease adverselyaffects the quality of life of its victims, with subject complaintsranging from unsightly nails and discomfort with footwear, to moreserious complications including secondary bacterial infections.

Many methods are known for the treatment of fungal infections, includingthe oral and topical use of antibiotics (e.g., nystatin and amphotericinB), imidazole anti-fungal agents such as miconazole, clotrimazole,fluconazole, econazole and sulconazole, and non-imidazole fungal agentssuch as the allylamine derivatives terbinafine and naftifine, and thebenzylamine butenafine.

However, onychomycosis has proven to be resistant to most treatments.Nail fungal infections reside in an area difficult to access byconventional topical treatment and anti-fungal drugs cannot readilypenetrate the nail plate to reach the infection sites under the nail.Therefore, onychomycosis has traditionally been treated by oraladministration of anti-fungal drugs; however, clearly this isundesirable due to the potential for side effects of such drugs, inparticular those caused by the more potent anti-fungal drugs such asitraconazole and ketoconazole. An alternative method of treatment ofonychomycosis is by removal of the nail before treating with a topicallyactive anti-fungal agent; such a method of treatment is equallyundesirable. Systemic antimycotic agents require prolonged use and havethe potential for significant side effects. Topical agents have usuallybeen of little benefit, primarily because of poor penetration of theanti-fungal agents into and through the nail mass.

In an exemplary embodiment, the invention provides a method of treatingor preventing onychomycosis. The method includes administering to ahuman or an animal a therapeutically effective amount of a compound ofthe invention, or a pharmaceutical formulation of the invention,sufficient to treat or prevent onychomycosis. In another exemplaryembodiment, the method includes administering the pharmaceuticalformulation of the invention at a site which is a member selected fromthe skin, nail, hair, hoof, claw and the skin surrounding the nail,hair, hoof and claw. In another exemplary embodiment, the pharmaceuticalformulation includes a compound described herein. The method includesadministering to a human or an animal a therapeutically effective amountof 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole, sufficient to treator prevent onychomycosis.

VI. a) 2) Other Unugjal and Periungual Infections

In an exemplary embodiment, the invention provides a method of treatingor preventing an ungual or periungual infection in a human or an animal.This method comprising administering to the human or the animal atherapeutically effective amount of a compound of the invention, therebytreating or preventing the ungual or periungual infection. In anexemplary embodiment, the ungual or periungual infection isonychomycosis. In an exemplary embodiment, the ungual or periungualinfection is a member selected from: onychomycosis, chloronychia,paronychias, erysipeloid, onychorrhexis, gonorrhea, swimming-poolgranuloma, larva migrans, leprosy, Orf nodule, milkers' nodules,herpetic whitlow, acute bacterial perionyxis, chronic perionyxis,sporotrichosis, syphilis, tuberculosis verrucosa cutis, tularemia,tungiasis, peri- and subungual warts, zona, nail dystrophy(trachyonychia), and dermatological diseases with an effect on thenails, such as psoriasis, pustular psoriasis, alopecia aerata,parakeratosis pustulosa, contact dermatosis, Reiter's syndrome,psoriasiform acral dermatitis, lichen planus, idiopathy atrophy in thenails, lichin nitidus, lichen striatus, inflammatory linear verrucousepidermal naevus (ILVEN), alopecia, pemphigus, bullous pemphigoid,acquired epidermolysis bullosa, Darier's disease, pityriasis rubrapilaris, palmoplantar keratoderma, contact eczema, polymorphic erythema,scabies, Bazex syndrome, systemic scleroderma, systemic lupuserythematosus, chronic lupus erythematosus, dermatomyositus.

The compounds and pharmaceutical formulations of the invention usefulfor ungual and periungual applications also find application in thecosmetics field, in particular for the treatment of irregularities ofthe nails, koilonychias, Beau's lines, longitudinal ridging, ingrownnails.

In an exemplary embodiment, the infection is of the skin, nail, hair,claw or hoof, hair, ear and eye and is a member selected fromSporotrichosis, Mycotic keratitis, Extension oculomycosis, Endogenousoculomycosis, Lobomycosis, Mycetoma, Piedra, Pityriasis versicolor,Tinea corporis, Tinea cruris, Tinea pedis, Tinea barbae, Tinea capitis,Tinea nigra, Otomycosis, Tinea favosa, Chromomycosis, and TineaImbricata. In an exemplary embodiment, the compound useful for treatingthese infections is 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole.

VI. b) Methods of Treating Systemic Diseases

In another aspect, the invention provides a method of treating asystemic disease. The method involves contacting an animal with acompound of the invention. The method of delivery for treatment ofsystemic disesases can be oral, intravenous, transdermal, inhalation,intraperitoneal, and subcutaneous. In an exemplary embodiment, thecompound administered is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole.

In an exemplary embodiment, the infection is systemic and is a memberselected from candidiasis, aspergillosis, coccidioidomycosis,cryptococcosis, histoplasmosis, blastomycosis, paracoccidioidomycosis,zygomycosis, phaeohyphomycosis and rhinosporidiosis.

VI. c) Methods of Treating Diseases Involving Viruses

The compounds of the invention are useful for the treatment of diseasesof both animals and humans, involving viruses. In an exemplaryembodiment, the disease is a member selected from hepatitis A-B-C,yellow fever, respiratory syncytial, influenza, AIDS, herpes simplex,chicken pox, varicella zoster, and Epstein-Barr disease.

VI. d) Methods of Treating Diseases Involving Parasites

The compounds of the invention are useful for the treatment of diseasesof both animals and humans, involving parasites. In an exemplaryembodiment, the disease is a member selected from malaria, Chagas'disease, Leishmaniasis, African sleeping sickness (African humantrypanosomiasis), giardiasis, toxoplasmosis, amebiasis andcryptosporidiosis.

In any of the methods according to the present invention set forthabove, it is preferred that the aminoacyl tRNA synthetase is anaminoacyl tRNA synthetase comprising an editing domain. The editingdomain is encoded by a portion of the aminoacyl tRNA synthetase involvedin proofreading. The editing domain is preferably encoded by a DNAportion having at least conserved residues compared after alignment withthe editing site of the leucyl-tRNA synthetase, valyl-tRNA synthetaseand isoleucyl-tRNA synthetase. More preferably the synthetase isselected from the group consisting of the valyl-tRNA synthetase,isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, alanyl-tRNAsynthetase, prolyl-tRNA synthetase, threonyl-tRNA synthetase,phenyl-tRNA synthetase and lysyl-tRNA synthetase which are known to havean editing site or domain (see for Ile RS Baldwin, A. N. and Berg, P.(1966) J. Biol. Chem. 241, 839-845 and Eldred, E. W. and Schimmel, P. R.(1972) J. Biol. Chem. 247, 2961-2964; for Val RS, Fersht, A. R. andKaethner, M. M. (1976) Biochemistry. 15 (15), 3342-3346; for Leu RS,English, S. et al., (1986) Nucleic Acids Research. 14 (19), 7529-7539;for Ala RS, Tsui, W. C. and Fersht, A. R. (1981) Nucleic Acids Research.9, 7529-7539; for Pro RS, Beuning, P. J. and Musier-Forsyth, K. (2000)PNAS. 97 (16), 8916-8920; for Thr RS, Sankaranarayanan, R. et al.,(2000) Nat. Struct. Biol. 7, 461-465 and Musier-Foryth, K. and Beuning,P. J. (2000) Nat. Struct. Biol. 7, 435-436; for PheRS, Yarus, M. (1972)PNAS. 69, 1915-1919 and for LysRS, Jakubowski, H. (1997) Biochemistry.36, 11077-11085.

VII. Methods of Nail Penetration

It is believed that poor penetration of the active agent through thehoof or nail plate and/or excessive binding to keratin, (the majorprotein in nails and hair) are the reasons for the poor efficacy of 8%ciclopirox w/w in commercial lacquer and other topical treatments thathave failed in clinical trials. In mild cases of onychomycosis, thepathogenic fungi reside in the nail plate only. In moderate to severecases the pathogenic fungi establish a presence in the nail plate and inthe nail bed. If the infection is cleared from the nail plate but notfrom the nail bed, the fungal pathogen can re-infect the nail plate.Therefore, to effectively treat onychomycosis, the infection must beeliminated from the nail plate and the nail bed. To do this, the activeagent must penetrate and disseminate substantially throughout the nailplate and nail bed.

It is believed that in order for an active agent to be effective oncedisseminated throughout the infected area, it must be bioavailable tothe fungal pathogen and cannot be so tightly bound to keratin that thedrug cannot inhibit growth or kill the infecting fungi.

An understanding of the morphology of the nail plate suggests certainphysicochemical properties of an active agent that would facilitatepenetration of the nail plate. The desired physicochemical propertiesare described throughout. The tested compounds of the present inventionare able to penetrate the nail plate and were also active againstTrichophyton rubrum and mentagrophytes and other species. In addition,the tested compounds are also active against Trichophyton rubrum in thepresence of 5% keratin powder.

In an exemplary embodiment, the invention provides a method of killingor inhibiting growth of a microorganism present in a human nail unit,wherein said human nail unit comprises a nail plate. The methodcomprising contacting a dorsal layer of the nail plate with a compoundcapable of penetrating the nail plate, traveling through the nail plateto a nail bed underlying said nail plate, and contacting saidmicroorganism, under conditions sufficient for said compound topenetrate said nail plate. In this embodiment, the compound has amolecular weight of between about 100 Da and about 200 Da, a log P valueof between about 1.0 and about 2.6, a water solubility greater thanabout 0.1 mg/mL octanol/saturated water, and an MIC of less than 16 g/mLagainst said microorganism, thereby killing or inhibiting the growth ofsaid microorganism.

In an exemplary embodiment, the compound has a structure according toFormula (I) described herein. In another exemplary embodiment, thecompound has a structure according to Formula (Ia)-(Iaa) describedherein. In another exemplary embodiment, the compound has a structureaccording to a member selected from Formula (I)-(Iaa), wherein R^(9a),R^(10a), R^(11a) and R^(12a) are members independently selected frommembers independently selected from H, halogen, cyano, nitro,substituted or unsubstituted methoxy, substituted or unsubstitutedmethyl, substituted or unsubstituted ethoxy, substituted orunsubstituted ethyl, trifluoromethyl, substituted or unsubstitutedhydroxymethyl, substituted or unsubstituted hydroxyalkyl, substituted orunsubstituted benzyl, substituted or unsubstituted phenyl, substitutedor unsubstituted phenyloxy, substituted or unsubstituted phenyl methoxy,substituted or unsubstituted thiophenyloxy, substituted or unsubstitutedpyridinyloxy, substituted or unsubstituted pyrimidinyloxy, substitutedor unsubstituted benzylfuran, substituted or unsubstituted methylthio,substituted or unsubstituted mercaptomethyl, substituted orunsubstituted mercaptoalkyl, substituted or unsubstituted phenylthio,substituted or unsubstituted thiophenylthio, substituted orunsubstituted phenyl methylthio, substituted or unsubstitutedpyridinylthio, substituted or unsubstituted pyrimidinylthio, substitutedor unsubstituted benzylthiofuranyl, substituted or unsubstitutedphenylsulfonyl, substituted or unsubstituted benzylsulfonyl, substitutedor unsubstituted phenylmethylsulfonyl, substituted or unsubstitutedthiophenylsulfonyl, substituted or unsubstituted pyridinylsulfonyl,substituted or unsubstituted pyrimidinylsulfonyl, substituted orunsubstituted sulfonamidyl, substituted or unsubstituted phenylsulfinyl,substituted or unsubstituted benzylsulfinyl, substituted orunsubstituted phenylmethylsulfinyl, substituted or unsubstitutedthiophenylsulfinyl, substituted or unsubstituted pyridinylsulfinyl,substituted or unsubstituted pyrimidinylsulfinyl, substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedtrifluoromethylamino, substituted or unsubstituted aminomethyl,substituted or unsubstituted alkylaminomethyl, substituted orunsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted benzylamino, substitutedor unsubstituted phenylamino, substituted or unsubstitutedthiophenylamino, substituted or unsubstituted pyridinylamino,substituted or unsubstituted pyrimidinylamino, substituted orunsubstituted indolyl, substituted or unsubstituted morpholino,substituted or unsubstituted alkylamido, substituted or unsubstitutedarylamido, substituted or unsubstituted ureido, substituted orunsubstituted carbamoyl, and substituted or unsubstituted piperizinyl.In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a)are members independently selected from H, fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy. In another exemplary embodiment, wherein R^(9a) is Hand R^(12a) is H. In another exemplary embodiment, the compound is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole.

In another exemplary embodiment, the invention provides a method oftreating a disease caused by a microorganism present in a human nailunit, wherein said human nail unit comprises a nail plate, said methodcomprising: contacting a dorsal layer of the nail plate with a compoundcapable of penetrating the nail plate, traveling through the nail plateto a nail bed underlying said nail plate, and contacting saidmicroorganism, under conditions sufficient for said compound topenetrate said nail plate and to treat said disease. In this embodiment,the compound has a molecular weight of between about 100 Da and about200 Da; a log P value of between about 1.0 and about 2.6; a watersolubility greater than about 0.1 mg/mL octanol/saturated water, and anMIC of less than 16 μg/mL against said microorganism, thereby treatingsaid disease. In an exemplary embodiment, the compound has a structureaccording to Formula (I) described herein. In another exemplaryembodiment, the compound has a structure which is a member selected fromFormula (Ia)-(Iaa) described herein.

In another aspect, the invention provides a method of delivering acompound from the dorsal layer of the nail plate to the nail bed. Thismethod comprises contacting the cell with a compound capable ofpenetrating the nail plate, under conditions sufficient to penetrate thenail. The compound has a molecular weight of between about 100 and about200 Da. The compound also has a log P value of between about 1.0 andabout 2.6. The compound additionally has a water solubility betweenabout 0.1 mg/mL and 1 g/mL octanol/saturated water, thereby deliveringsaid compound.

In a preferred embodiment, the physicochemical properties of thecompound of the invention, described by quantities predictive formigration of the compound through the nail plate, including, but notlimited to, molecular weight, log P and solubility in water, and thelike, are effective to provide substantial penetration of the nailplate.

Compounds with a molecular weight of less than 200 Da penetrate the nailplate in a manner superior to the commercially available treatment foronychomycosis. In one embodiment of the present invention the compoundhas a molecular weight of between 130 and 200. In another embodiment ofthis invention, the compound has a molecular weight of from about 140 toabout 200 Da. In another embodiment of this invention, the compound hasa molecular weight of from about 170 to about 200 Da. In anotherembodiment of this invention, the compound has a molecular weight offrom about 155 to about 190 Da. In another embodiment of this invention,the compound has a molecular weight of from about 165 to about 185 Da.In another embodiment of this invention, the compound has a molecularweight of from about 145 to about 170 Da. In yet another embodiment themolecular weight is either 151.93 or 168.39 Da.

In one embodiment of the present invention the compound has a log Pvalue of between about −3.5 to about 2.5. In another exemplaryembodiment, the compound has a log P value of from about −1.0 to about2.5. In another exemplary embodiment, the compound has a log P value offrom about −1.0 to about 2.0. In another exemplary embodiment, thecompound has a log P value of from about −0.5 to about 2.5. In anotherexemplary embodiment, the compound has a log P value of from about −0.5to about 1.5. In another exemplary embodiment, the compound has a log Pvalue of from about 0.5 to about 2.5. In another exemplary embodiment,the compound has a log P value of from about 1.0 to about 2.5. In yetanother exemplary embodiment, the compound has a log P value of 1.9 or2.3.

Also contemplated by the present invention is a compound with a log Pvalue less then 2.5, with a molecular weight less than 200 Da, that arestill able to penetrate the nail plate.

In one embodiment of the present invention the compound has a watersolubility between about 0.1 mg/mL to 1 g/mL in octanol saturated water.In one embodiment of the present invention the compound has a watersolubility of between 0.1 mg/mL and 100 mg/mL. In another embodiment ofthis invention, the compound has a water solubility of from about 0.1mg/mL and 10 mg/mL. In another embodiment of this invention, thecompound has a water solubility of from about 0.1 mg/mL and 1 mg/mL. Inanother embodiment of this invention, the compound has a watersolubility of from about 5 mg/mL and 1 g/mL. In another embodiment ofthis invention, the compound has a water solubility of from about 10mg/mL and 500 g/mL. In another embodiment of this invention, thecompound has a water solubility of from about 80 mg/mL and 250 mg/mL.

In an exemplary embodiment, the present invention provides a compoundwith a log P value selected from a range above, with a molecular weightselected from a range above, that are still able to penetrate the nailplate.

In an exemplary embodiment, the present invention provides compoundswith a molecular weight selected from a range above, with a watersolubility selected from a range above, that are still able to penetratethe nail plate.

In an exemplary embodiment, the present invention provides compoundswith a log P selected from a range above, with a water solubilityselected from a range above, that are still able to penetrate the nailplate.

In an exemplary embodiment, the present invention provides compoundswith a molecular weight selected from a range above, with a log Pselected from a range above, and with a water solubility selected from arange above, that are still able to penetrate the nail plate.

Penetration of the nail by the active ingredient may be effected by thepolarity of the formulation. However, the polarity of the formulation isnot expected have as much influence on nail penetration as some of theother factors, such as the molecular weight or the log P of the activeingredient. The presence of penetration enhancing agents in theformulation is likely to increase penetration of the active agent whencompared to similar formulations containing no penetration enhancingagent.

Some examples of molecules with optimal physicochemical properties aregiven in the table below.

                Structure:

  (compound 1)

  (compound 2) Formula: C₇H₆BFO₂ C₇H₆BClO₂ Molecular 151.93 168.39weight (Da): Plasma protein 66 83 binding (%): LogP: 1.9 2.3 Watersolubility >100 >100 (μg/mL):

Compound 3 below is an example of a compound similar in molecular weightto ciclopirox, and like ciclopirox, penetrates the nail plate poorly.

                        Structure:

  (compound 3) Formula: C₁₃H₁₀BFO Molecular weight (Da): 212.03 Plasmaprotein binding (%): 100   cLogP:  3.55 Water solubility (μg/mL): notdetermined

In a preferred embodiment the topical formulations including a compounddescribed herein has a total molecular weight of less than 200 Da, has aLog P of less than 2.5, and a minimum inhibitory concentration againstTrichophyton rubrum that is substantially unchanged in the presence of5% keratin.

The efficacy coefficient (defined as flux over MIC) of a compound alsoinforms one of skill regarding whether the compound may be effective inkilling a microorganism, inhibiting the growth of a microorganism, ortreating a disease which is caused by a microorganism present in a humannail unit, wherein said human nail unit comprises a nail plate. Themethod comprises: contacting a dorsal layer of the nail plate with acompound capable of penetrating the nail plate, traveling through thenail plate to a nail bed underlying said nail plate, and contacting saidmicroorganism, under conditions sufficient for said compound topenetrate said nail plate and to treat said disease, wherein thecompound has an efficacy coefficient above 10.

In an exemplary embodiment, the compound has an efficacy coefficientbetween about 10 and about 1000. In an exemplary embodiment, thecompound has an efficacy coefficient between about 30 and about 100. Inan exemplary embodiment, the compound has an efficacy coefficientbetween about 100 and about 500. In an exemplary embodiment, thecompound has an efficacy coefficient between about 25 and about 200.

This invention is still further directed to methods for treating afungal infection mediated at least in part by dermatophytes,Trichophyton, Microsporum or Epidermophyton species, or a yeast-likefungi including Candida species, in a human or an animal, which methodscomprise administering to a human or an animal, that has been diagnosedwith said fungal infection or is at risk of developing said fungalinfection, a pharmaceutical composition comprising a pharmaceuticallyacceptable diluent and a therapeutically effective amount of a compounddescribed herein or mixtures of one or more of such compounds. In oneembodiment the infection is onychomycosis.

Compounds contemplated by the present invention may have broad spectrumantifungal activity and as such may be candidates for use against othercutaneous fungal infections.

The methods provided in this aspect of the invention are useful in thepenetration of nails and hoofs, as well as the treatment of ungual andperiungual conditions.

VIII. Pharmaceutical Formulations

In another aspect, the invention is a pharmaceutical formulation whichincludes: (a) a pharmaceutically acceptable excipient; and (b) acompound of the invention. In another aspect, the invention is apharmaceutical formulation which includes: (a) a pharmaceuticallyacceptable excipient; and (b) a compound having a structure which is amember selected from Formulae (I), (Ia), (Ib), (Ic), (Id) (Ie), (If),(Ig), (Ih) (Ii), (Ij), (Ik), (Il) (Im), (In), (Io), (Ip) (Iq), (Ir),(Is), (It), (Iu), (Iv), (Iw), (Ix) (Iy), (Iz), (Iaa), (Iab), (Iac),(Iad), (Iae), (Iaf), (Iag), (Iah), (Iai), (Iaj), (Iak), (II), (IIa),(IIb), (IIc), (IId), (III). In another aspect, the invention is apharmaceutical formulation which includes: (a) a pharmaceuticallyacceptable excipient; and (b) a compound which has a structure accordingto Formulae (VIII), (VIIIa), (VIIIb), (VIIIc), (VIIId), (VIIIe). Inanother aspect, the invention is a pharmaceutical formulation whichincludes: (a) a pharmaceutically acceptable excipient; and (b) acompound which has a structure which is a member selected from D1-D19,E1-E19, (VIII), (VIIIa), (VIIIb), (VIIIc), (VIIId), (VIIIe). In anotheraspect, the invention is a pharmaceutical formulation which includes:(a) a pharmaceutically acceptable excipient; and (b) a acyclic boronicester of the invention. In an exemplary embodiment, the compound isdescribed in FIG. 19. In another exemplary embodiment, the compound isdescribed in FIG. 20. In another exemplary embodiment, the invention isa pharmaceutical formulation which includes: (a) a pharmaceuticallyacceptable excipient; and (b) a acyclic boronic ester of the invention.

In another aspect, the invention is a pharmaceutical formulationcomprising: (a) a pharmaceutically acceptable excipient; and (b) acompound having a structure according to Formula I:

wherein B is boron. R^(1a) is a member selected from a negative charge,a salt counterion, H, cyano, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl. M isa member selected from oxygen, sulfur and NR^(2a). R^(2a) is a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. J is amember selected from (CR^(3a)R^(4a))_(n1) and CR^(5a). R^(3a), R^(4a),and R^(5a) are members independently selected from H, halogen, cyano,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. The index n1 is an integerselected from 0 to 2. W is a member selected from C═O (carbonyl),(CR^(6a)R^(7a))_(m1) and CR^(8a). R^(6a), R^(7a), and R^(8a) are membersindependently selected from H, halogen, cyano, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. The index m1 is an integer selected from 0and 1. A is a member selected from CR^(9a) and N. D is a member selectedfrom CR^(10a) and N. E is a member selected from CR^(11a) and N. G is amember selected from CR^(12a) and N. R^(9a), R^(10a), R^(11a) andR^(12a) are members independently selected from H, OR*, NR*R**, SR*,—S(O)R*, —S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, nitro,halogen, cyano, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. Each R*and R** are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. The combination of nitrogens (A+D+E+G) is aninteger selected from 0 to 3. A member selected from R^(3a), R^(4a) andR^(5a) and a member selected from R^(6a), R^(7a) and R^(8a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring. R^(3a) and R^(4a), together with the atoms towhich they are attached, are optionally joined to form a 4 to 7 memberedring. R^(6a) and R^(7a), together with the atoms to which they areattached, are optionally joined to form a 4 to 7 membered ring. R^(9a)and R^(10a), together with the atoms to which they are attached, areoptionally joined to form a 4 to 7 membered ring. R^(10a) and R^(1a),together with the atoms to which they are attached, are optionallyjoined to form a 4 to 7 membered ring. R^(11a) and R^(12a), togetherwith the atoms to which they are attached, are optionally joined to forma 4 to 7 membered ring.

In another exemplary embodiment, there is a proviso that the compoundcannot have a structure according to Formula (Ix):

wherein R^(7b) is a member selected from H, methyl, ethyl and phenyl.R^(10b) is a member selected from H, OH, NH₂, SH, halogen, substitutedor unsubstituted phenoxy, substituted or unsubstituted phenylalkyloxy,substituted or unsubstituted phenylthio and substituted or unsubstitutedphenylalkylthio. R^(11b) is a member selected from H, OH, NH₂, SH,methyl, substituted or unsubstituted phenoxy, substituted orunsubstituted phenylalkyloxy, substituted or unsubstituted phenylthioand substituted or unsubstituted phenylalkylthio. In another exemplaryembodiment, there is a proviso that the compound cannot have a structureaccording to Formula (Ix) wherein R^(1b) is a member selected from anegative charge, H and a salt counterion. In another exemplaryembodiment, there is a proviso that the compound cannot have a structureaccording to Formula (Ix) wherein R^(10b) and R^(11b) are H. In anotherexemplary embodiment, there is a proviso that the compound cannot have astructure according to Formula (Ix) wherein one member selected fromR^(10b) and R^(11b) is H and the other member selected from R^(10b) andR^(11b) is a member selected from halo, methyl, cyano, methoxy,hydroxymethyl and p-cyanophenyloxy. In another exemplary embodiment,there is a proviso that the compound cannot have a structure accordingto Formula (Ix) wherein R^(10b) and R^(11b) are members independentlyselected from fluoro, chloro, methyl, cyano, methoxy, hydroxymethyl, andp-cyanophenyl. In another exemplary embodiment, there is a proviso thatthe compound cannot have a structure according to Formula (Ix) whereinR^(1b) is a member selected from a negative charge, H and a saltcounterion; R^(7b) is H; R^(10b) is F and R^(11b) is H. In anotherexemplary embodiment, there is a proviso that the compound cannot have astructure according to Formula (Ix) wherein R^(11b) and R^(12b), alongwith the atoms to which they are attached, are joined to form a phenylgroup. In another exemplary embodiment, there is a proviso that thecompound cannot have a structure according to Formula (Ix) whereinR^(1b) is a member selected from a negative charge, H and a saltcounterion; R^(7b) is H; R^(10b) is 4-cyanophenoxy; and R^(11b) is H.

In another exemplary embodiment, there is a proviso that the compoundcannot have a structure according to Formula (Iy)

wherein R^(10b) is a member selected from H, halogen, CN and substitutedor unsubstituted C₁₋₄ alkyl.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure according to Formula (Ia):

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberindependently selected from H, cyano, substituted or unsubstitutedmethyl, substituted or unsubstituted ethyl, trifluoromethyl, substitutedor unsubstituted hydroxymethyl, substituted or unsubstitutedhydroxyalkyl, substituted or unsubstituted benzyl, substituted orunsubstituted phenyl, substituted or unsubstituted mercaptomethyl,substituted or unsubstituted mercaptoalkyl, substituted or unsubstitutedaminomethyl, substituted or unsubstituted alkylaminomethyl, substitutedor unsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted indolyl and substituted orunsubstituted amido. In another exemplary embodiment, each R^(3a) andR^(4a) is a member independently selected from cyano, substituted orunsubstituted methyl, substituted or unsubstituted ethyl,trifluoromethyl, substituted or unsubstituted hydroxymethyl, substitutedor unsubstituted hydroxyalkyl, substituted or unsubstituted benzyl,substituted or unsubstituted phenyl, substituted or unsubstitutedmercaptomethyl, substituted or unsubstituted mercaptoalkyl, substitutedor unsubstituted aminomethyl, substituted or unsubstitutedalkylaminomethyl, substituted or unsubstituted dialkylaminomethyl,substituted or unsubstituted arylaminomethyl, substituted orunsubstituted indolyl, substituted or unsubstituted amido.

In another exemplary embodiment, each R^(3a) and R^(4a) is a memberselected from H, substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted propyl, substituted orunsubstituted isopropyl, substituted or unsubstituted butyl, substitutedor unsubstituted t-butyl, substituted or unsubstituted phenyl andsubstituted or unsubstituted benzyl. In another exemplary embodiment,R^(3a) and R^(4a) is a member selected from methyl, ethyl, propyl,isopropyl, butyl, t-butyl, phenyl and benzyl. In another exemplaryembodiment, R^(3a) is H and R^(4a) is a member selected from methyl,ethyl, propyl, isopropyl, butyl, t-butyl, phenyl and benzyl. In anotherexemplary embodiment, R^(3a) is H and R^(4a)H.

In another exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) isa member independently selected from H, OR*, NR*R**, SR*, —S(O)R*,—S(O)₂R*, —S(O)₂NR*R**, —C(O)R*, —C(O)OR*, —C(O)NR*R**, halogen, cyano,nitro, substituted or unsubstituted methoxy, substituted orunsubstituted methyl, substituted or unsubstituted ethoxy, substitutedor unsubstituted ethyl, trifluoromethyl, substituted or unsubstitutedhydroxymethyl, substituted or unsubstituted hydroxyalkyl, substituted orunsubstituted benzyl, substituted or unsubstituted phenyl, substitutedor unsubstituted phenyloxy, substituted or unsubstituted phenyl methoxy,substituted or unsubstituted thiophenyloxy, substituted or unsubstitutedpyridinyloxy, substituted or unsubstituted pyrimidinyloxy, substitutedor unsubstituted benzylfuran, substituted or unsubstituted methylthio,substituted or unsubstituted mercaptomethyl, substituted orunsubstituted mercaptoalkyl, substituted or unsubstituted phenylthio,substituted or unsubstituted thiophenylthio, substituted orunsubstituted phenyl methylthio, substituted or unsubstitutedpyridinylthio, substituted or unsubstituted pyrimidinylthio, substitutedor unsubstituted benzylthiofuranyl, substituted or unsubstitutedphenylsulfonyl, substituted or unsubstituted benzylsulfonyl, substitutedor unsubstituted phenylmethylsulfonyl, substituted or unsubstitutedthiophenylsulfonyl, substituted or unsubstituted pyridinylsulfonyl,substituted or unsubstituted pyrimidinylsulfonyl, substituted orunsubstituted sulfonamidyl, substituted or unsubstituted phenylsulfinyl,substituted or unsubstituted benzylsulfinyl, substituted orunsubstituted phenylmethylsulfinyl, substituted or unsubstitutedthiophenylsulfinyl, substituted or unsubstituted pyridinylsulfinyl,substituted or unsubstituted pyrimidinylsulfinyl, substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedtrifluoromethylamino, substituted or unsubstituted aminomethyl,substituted or unsubstituted alkylaminomethyl, substituted orunsubstituted dialkylaminomethyl, substituted or unsubstitutedarylaminomethyl, substituted or unsubstituted benzylamino, substitutedor unsubstituted phenylamino, substituted or unsubstitutedthiophenylamino, substituted or unsubstituted pyridinylamino,substituted or unsubstituted pyrimidinylamino, substituted orunsubstituted indolyl, substituted or unsubstituted morpholino,substituted or unsubstituted alkylamido, substituted or unsubstitutedarylamido, substituted or unsubstituted ureido, substituted orunsubstituted carbamoyl, and substituted or unsubstituted piperizinyl.In an exemplary embodiment, R^(9a), R^(10a), R^(11a) and R^(12a) areselected from the previous list of substituents with the exception of—C(O)R*, —C(O)OR*, —C(O)NR*R**.

In another exemplary embodiment, R^(6a), R^(7a), R^(9a), R^(10a),R^(11a) and R^(12a) are members independently selected from fluoro,chloro, bromo, nitro, cyano, amino, methyl, hydroxylmethyl,trifluoromethyl, methoxy, trifluoromethyoxy, ethyl, diethylcarbamoyl,pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidinyl, piperizino,piperizinyl, piperizinocarbonyl, piperizinylcarbonyl, carboxyl,1-tetrazolyl, 1-ethoxycarbonylmethoxy, carboxymethoxy, thiophenyl,3-(butylcarbonyl)phenylmethoxy, 1H-tetrazol-5-yl,1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-, 1-ethoxycarbonyl-,carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-, thiophen-3-yl,thiophen-3-ylthio, 4-fluorophenylthio, butylcarbonylphenylmethoxy,butylcarbonylphenylmethyl, butylcarbonylmethyl,1-(piperidin-1-yl)carbonyl)methyl, 1-(piperidin-1-yl)carbonyl)methoxy,1-(piperidin-2-yl)carbonyl)methoxy, 1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy,4-fluorobenzyloxy, unsubstituted phenyl, unsubstituted benzyl.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure which is a member according the followingformulas:

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure according to one of Formulae I-Io withsubstituent selections for R^(9a), R^(10a), R^(11a) and R^(12a)including all the possibilities contained in paragraph 106 except for H.In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure according to one of Formulae Ib-Io withsubstituent selections for R^(9a), R^(10a), R^(11a) and R^(12a)including all the possibilities contained in paragraph 107 except for H.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a formula according to Formulae (Ib)-(Ie) whereinR^(1a) is a member selected from H, a negative charge and a saltcounterion and the remaining R group (R^(9a) in Ib, R^(10a) in Ic,R^(11a) in Id, and R^(12a) in Ie) is a member selected from fluoro,chloro, bromo, nitro, cyano, amino, methyl, hydroxylmethyl,trifluoromethyl, methoxy, trifluoromethyoxy, ethyl, diethylcarbamoyl,pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidinyl, piperizino,piperizinyl, piperizinocarbonyl, piperizinylcarbonyl, carboxyl,1-tetrazolyl, 1-ethoxycarbonylmethoxy, carboxymethoxy, thiophenyl,3-(butylcarbonyl)phenylmethoxy, 1H-tetrazol-5-yl,1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-, 1-ethoxycarbonyl-,carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-, thiophen-3-yl,thiophen-3-ylthio, 4-fluorophenylthio, butylcarbonylphenylmethoxy,butylcarbonylphenylmethyl, butylcarbonylmethyl,1-(piperidin-1-yl)carbonyl)methyl, 1-(piperidin-1-yl)carbonyl)methoxy,1-(piperidin-2-yl)carbonyl)methoxy, 1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy and4-fluorobenzyloxy.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a formula according to Formulae (If)-(Ik) whereinR^(1a) is a member selected from H, a negative charge and a saltcounterion and each of the remaining two R groups (R^(9a) and R^(10a) inIf, R^(9a) and R^(11a) in Ig, R^(9a) and R^(12a) in Ih, Ro^(10a) andR^(11a) in Ii, R^(10a) and R^(12a) in Ij, R^(11a) and R^(12a) in Ik) isa member independently selected from fluoro, chloro, bromo, nitro,cyano, amino, methyl, hydroxylmethyl, trifluoromethyl, methoxy,trifluoromethyoxy, ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl,pyridin-4-yl, pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a formula according to Formulae (Il)-(Io) whereinR^(1a) is a member selected from H, a negative charge and a saltcounterion and each of the remaining three R groups (R^(9a), R^(10a),R^(11a) in (Il), R^(9a), R^(10a), R^(12a) in (Im), R^(9a), R^(11a),R^(12a) in (In), R^(10a), R^(11a), R^(12a) in (Io)) is a memberindependently selected from fluoro, chloro, bromo, nitro, cyano, amino,methyl, hydroxylmethyl, trifluoromethyl, methoxy, trifluoromethyoxy,ethyl, diethylcarbamoyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl,pyrimidinyl, piperizino, piperizinyl, piperizinocarbonyl,piperizinylcarbonyl, carboxyl, 1-tetrazolyl, 1-ethoxycarbonylmethoxy,carboxymethoxy, thiophenyl, 3-(butylcarbonyl)phenylmethoxy,1H-tetrazol-5-yl, 1-ethoxycarbonylmethyloxy-, 1-ethoxycarbonylmethyl-,1-ethoxycarbonyl-, carboxymethoxy-, thiophen-2-yl, thiophen-2-ylthio-,thiophen-3-yl, thiophen-3-ylthio, 4-fluorophenylthio,butylcarbonylphenylmethoxy, butylcarbonylphenylmethyl,butylcarbonylmethyl, 1-(piperidin-1-yl)carbonyl)methyl,1-(piperidin-1-yl)carbonyl)methoxy, 1-(piperidin-2-yl)carbonyl)methoxy,1-(piperidin-3-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methoxy,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl)methyl,1-(4-(pyrimidin-2-yl)piperazin-1-yl)carbonyl,1-4-(pyrimidin-2-yl)piperazin-1-yl,1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl),1-(4-(pyridin-2-yl)piperazin-1-yl)carbonylmethyl,(1-(4-(pyridin-2-yl)piperazin-1-yl)carbonyl)-methoxy),1-(4-(pyridin-2-yl)piperazin-1-yl, 1H-indol-1-yl, morpholino-,morpholinyl, morpholinocarbonyl, morpholinylcarbonyl, phenylureido,phenylcarbamoyl, acetamido, 3-(phenylthio)-1H-indol-1-yl,3-(2-cyanoethylthio)-1H-indol-1-yl, benzylamino,5-methoxy-3-(phenylthio)-1H-indol-1-yl,5-methoxy-3-(2-cyanoethylthio)-1H-indol-1-yl)), 5-chloro-1H-indol-1-yl,5-chloro-3-(2-cyanoethylthio)-1H-indol-1-yl)), dibenzylamino,benzylamino, 5-chloro-3-(phenylthio)-1H-indol-1-yl)),4-(1H-tetrazol-5-yl)phenoxy, 4-(1H-tetrazol-5-yl)phenyl,4-(1H-tetrazol-5-yl)phenylthio, 2-cyanophenoxy, 3-cyanophenoxy,4-cyanophenoxy, 2-cyanophenylthio, 3-cyanophenylthio, 4-cyanophenylthio,2-chlorophenoxy, 3-chlorophenoxy, 4-chlorophenoxy, 2-fluorophenoxy,3-fluorophenoxy, 4-fluorophenoxy, 2-cyanobenzyloxy, 3-cyanobenzyloxy,4-cyanobenzyloxy, 2-chlorobenzyloxy, 3-chlorobenzyloxy,4-chlorobenzyloxy, 2-fluorobenzyloxy, 3-fluorobenzyloxy, and4-fluorobenzyloxy.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that is a member selected from:

In an exemplary embodiment, the compound of the invention has astructure which is a member selected from:

in which q is a number between 0 and 1. R^(g) is halogen. R^(a), R^(b),R^(c), R^(d) and R^(e) are members independently selected from a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. In anexemplary embodiment, the compound in the pharmaceutical formulation isa member selected from

In an exemplary embodiment, the compound has a structure is a memberselected from:

In an exemplary embodiment, R^(a), R^(d) and R^(e) are each membersindependently selected from:

In an exemplary embodiment, R^(b) and R^(c) are members independentlyselected from H, methyl,

In another exemplary embodiment, R^(b) is H and R^(c) is a memberselected from H, methyl,

In another exemplary embodiment, R^(b) and R^(c) are, together with thenitrogen to which they are attached, optionally joined to form a memberselected from

In an exemplary embodiment, R^(a) is a member selected from

In an exemplary embodiment, R^(d) is a member selected from

In an exemplary embodiment, R^(e) is a member selected from

In another exemplary embodiment, the pharmaceutical formulationsdescribed herein can form a hydrate with water, a solvate with analcohol (e.g. methanol, ethanol, propanol); an adduct with an aminocompound (e.g. ammonia, methylamine, ethylamine); an adduct with an acid(e.g. formic acid, acetic acid); complexes with ethanolamine, quinoline,amino acids, and the like.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure according to Formula (Ip):

in which R^(x2) is a member selected from substituted or unsubstitutedC₁-C₅ alkyl and substituted or unsubstituted C₁-C₅ heteroalkyl. R^(y2)and R^(z2) are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure according to Formula (Iq):

wherein B is boron. R^(x2) is a member selected from substituted orunsubstituted C₁-C₅ alkyl and substituted or unsubstituted C₁-C₅heteroalkyl. R^(y2) and R^(z2) are members independently selected fromH, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. In another exemplaryembodiment, at least one member selected from R^(3a), R^(4a), R^(5a),R^(6a), R^(7a), R^(5a), R^(9a), R^(10a), R^(11a) and R^(12a) is a memberselected from nitro, cyano and halogen.

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure which is a member selected from thefollowing Formulae:

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a formula according to Formulae (Ib)-(Ie) wherein atleast one member selected from R^(3a), R^(4a), R^(5a), R^(6a), R^(7a),R^(8a), R^(9a), R^(10a), R^(11a) and R^(12a) is a member selected fromnitro, cyano, fluoro, chloro, bromo and cyanophenoxy. In an exemplaryembodiment, the pharmaceutical formulation comprises a compound that hasa structure which is a member selected from

In an exemplary embodiment, the pharmaceutical formulation comprises acompound that has a structure that is a member selected from

In another exemplary embodiment, there is a proviso that thepharmaceutical formulation cannot comprise a structure according toFormula (Iaa):

wherein R^(6b), R^(9b), R^(10b), R^(11b) and R^(12b) have the samesubstituent listings as described for Formulae (Ix) and (Iy) above.

The pharmaceutical formulations of the invention can take a variety offorms adapted to the chosen route of administration. Those skilled inthe art will recognize various synthetic methodologies that may beemployed to prepare non-toxic pharmaceutical formulations incorporatingthe compounds described herein. Those skilled in the art will recognizea wide variety of non-toxic pharmaceutically acceptable solvents thatmay be used to prepare solvates of the compounds of the invention, suchas water, ethanol, propylene glycol, mineral oil, vegetable oil anddimethylsulfoxide (DMSO).

The compositions of the invention may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. It is further understoodthat the best method of administration may be a combination of methods.Oral administration in the form of a pill, capsule, elixir, syrup,lozenge, troche, or the like is particularly preferred. The termparenteral as used herein includes subcutaneous injections, intradermal,intravascular (e.g., intravenous), intramuscular, spinal, intrathecalinjection or like injection or infusion techniques.

The pharmaceutical formulations containing compounds of the inventionare preferably in a form suitable for oral use, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to anymethod known in the art for the manufacture of pharmaceuticalformulations, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets may containthe active ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;and dispersing or wetting agents, which may be a naturally-occurringphosphatide, for example, lecithin, or condensation products of analkylene oxide with fatty acids, for example polyoxyethylene stearate,or condensation products of ethylene oxide with long chain aliphaticalcohols, for example heptadecaethyleneoxycetanol, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand a hexitol such as polyoxyethylene sorbitol monooleate, orcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides, for example polyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or morepreservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical formulations of the invention may also be in the form ofoil-in-water emulsions and water-in-oil emulsions. The oily phase may bea vegetable oil, for example olive oil or arachis oil, or a mineral oil,for example liquid paraffin or mixtures of these. Suitable emulsifyingagents may be naturally-occurring gums, for example gum acacia or gumtragacanth; naturally-occurring phosphatides, for example soy bean,lecithin, and esters or partial esters derived from fatty acids andhexitol; anhydrides, for example sorbitan monooleate; and condensationproducts of the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, and flavoring and coloringagents. The pharmaceutical formulations may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents, which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The composition of the invention may also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

Alternatively, the compositions can be administered parenterally in asterile medium. The drug, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

For administration to non-human animals, the composition containing thetherapeutic compound may be added to the animal's feed or drinkingwater. Also, it will be convenient to formulate animal feed and drinkingwater products so that the animal takes in an appropriate quantity ofthe compound in its diet. It will further be convenient to present thecompound in a composition as a premix for addition to the feed ordrinking water. The composition can also added as a food or drinksupplement for humans.

Dosage levels of the order of from about 5 mg to about 250 mg perkilogram of body weight per day and more preferably from about 25 mg toabout 150 mg per kilogram of body weight per day, are useful in thetreatment of the above-indicated conditions. The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the condition being treatedand the particular mode of administration. Dosage unit forms willgenerally contain between from about 1 mg to about 500 mg of an activeingredient.

Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most disorders, adosage regimen of 4 times daily or less is preferred. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

Preferred compounds of the invention will have desirable pharmacologicalproperties that include, but are not limited to, oral bioavailability,low toxicity, low serum protein binding and desirable in vitro and invivo half-lives. Penetration of the blood brain barrier for compoundsused to treat CNS disorders is necessary, while low brain levels ofcompounds used to treat peripheral disorders are often preferred.

Assays may be used to predict these desirable pharmacologicalproperties. Assays used to predict bioavailability include transportacross human intestinal cell monolayers, including Caco-2 cellmonolayers. Toxicity to cultured hepatocycles may be used to predictcompound toxicity. Penetration of the blood brain barrier of a compoundin humans may be predicted from the brain levels of laboratory animalsthat receive the compound intravenously.

Serum protein binding may be predicted from albumin binding assays. Suchassays are described in a review by Oravcova, et al. (Journal ofChromatography B (1996) volume 677, pages 1-27).

Compound half-life is inversely proportional to the frequency of dosageof a compound. In vitro half-lives of compounds may be predicted fromassays of microsomal half-life as described by Kuhnz and Gieschen (DrugMetabolism and Disposition, (1998) volume 26, pages 1120-1127).

The amount of the composition required for use in treatment will varynot only with the particular compound selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will ultimately be at the discretion ofthe attendant physician or clinician.

In an exemplary embodiment, the pharmaceutical formulation excipientcomprises ethanol and the pharmaceutical formulation compound is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In another exemplaryembodiment, the pharmaceutical formulation excipient comprises propyleneglycol and the pharmaceutical formulation compound is1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In an exemplaryembodiment the pharmaceutical formulation comprises: about propyleneglycol:ethanol 1:4, with 1:10 wt/volume of1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In an exemplaryembodiment the pharmaceutical formulation comprises: about 70% ethanol;about 20% poly(vinyl methyl ether-alt-maleic acid monobutyl ester);about 10% 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In anexemplary embodiment the pharmaceutical formulation comprises: about 56%ethanol; about 14% water; about 15% poly(2-hydroxyethyl methacrylate);about 5% dibutyl sebacate; about 10%1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. In an exemplaryembodiment the pharmaceutical formulation comprises: about 55% ethanol;about 15% ethyl acetate; about 15% poly(vinyl acetate); about 5% dibutylsebacate; about 10% 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole. Inanother exemplary embodiment,1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole is present in apharmaceutical formulation in a concentration which is a member selectedfrom 1%, 2.5%, 5%, 7.5%, 10% and 15% w/v. In another exemplaryembodiment, the pharmaceutical formulation is a lacquer.

In an exemplary embodiment, the pharmaceutical formulation excipientcomprises ethanol and the pharmaceutical formulation compound is5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anotherexemplary embodiment, the pharmaceutical formulation excipient comprisespropylene glycol and the pharmaceutical formulation compound is5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anexemplary embodiment the pharmaceutical formulation comprises: about 20%propylene glycol; about 70% ethanol; about 10%5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anexemplary embodiment the pharmaceutical formulation comprises: about 70%ethanol; about 20% poly(vinyl methyl ether-alt-maleic acid monobutylester); about 10%5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anexemplary embodiment the pharmaceutical formulation comprises: about 56%ethanol; about 14% water; about 15% poly(2-hydroxyethyl methacrylate);about 5% dibutyl sebacate; about 10%5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anexemplary embodiment the pharmaceutical formulation comprises: about 55%ethanol; about 15% ethyl acetate; about 15% poly(vinyl acetate); about5% dibutyl sebacate; about 10%5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole. In anotherexemplary embodiment,5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole is present ina pharmaceutical formulation in a concentration which is a memberselected from 1%, 2.5%, 5%, 7.5%, 10% and 15% w/v. In another exemplaryembodiment, the pharmaceutical formulation is a lacquer.

In an exemplary embodiment, the pharmaceutical formulation excipientcomprises ethanol and the pharmaceutical formulation compound is acompound described herein. In another exemplary embodiment, thepharmaceutical formulation excipient comprises propylene glycol and thepharmaceutical formulation compound is a compound described herein. Inan exemplary embodiment the pharmaceutical formulation comprises: about20% propylene glycol; about 70% ethanol; about 10% of a compounddescribed herein. In an exemplary embodiment the pharmaceuticalformulation comprises: about 70% ethanol; about 20% poly(vinyl methylether-alt-maleic acid monobutyl ester); about 10% of a compounddescribed herein. In an exemplary embodiment the pharmaceuticalformulation comprises: about 56% ethanol; about 14% water; about 15%poly(2-hydroxyethyl methacrylate); about 5% dibutyl sebacate; about 10%of a compound described herein. In an exemplary embodiment thepharmaceutical formulation comprises: about 55% ethanol; about 15% ethylacetate; about 15% poly(vinyl acetate); about 5% dibutyl sebacate; about10% of a compound described herein. In another exemplary embodiment, acompound described herein is present in a pharmaceutical formulation ina concentration which is a member selected from 1%, 2.5%, 5%, 7.5%, 10%and 15% w/v. In another exemplary embodiment, the pharmaceuticalformulation is a lacquer.

VII. a) Topical Formulations

In a preferred embodiment, the methods of the invention can be usedemployed through the topical application of the compounds describedherein.

The compositions of the present invention comprises fluid or semi-solidvehicles that may include but are not limited to polymers, thickeners,buffers, neutralizers, chelating agents, preservatives, surfactants oremulsifiers, antioxidants, waxes or oils, emollients, sunscreens, and asolvent or mixed solvent system. The solvent or mixed solvent system isimportant to the formation because it is primarily responsible fordissolving the drug. The best solvent or mixed solvent systems are alsocapable of maintaining clinically relevant levels of the drug insolution despite the addition of a poor solvent to the formulation. Thetopical compositions useful in the subject invention can be made into awide variety of product types. These include, but are not limited to,lotions, creams, gels, sticks, sprays, ointments, pastes, foams,mousses, and cleansers. These product types can comprise several typesof carrier systems including, but not limited to particles,nanoparticles, and liposomes. If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar or alginicacid or a salt thereof such as sodium alginate. Techniques forformulation and administration can be found in Remington: The Scienceand Practice of Pharmacy, supra. The formulation can be selected tomaximize delivery to a desired target site in the body.

Lotions, which are preparations that are to be applied to the skin,nail, hair, claw or hoof surface without friction, are typically liquidor semi-liquid preparations in which finely divided solid, waxy, orliquid are dispersed. Lotions will typically contain suspending agentsto produce better dispersions as well as compounds useful for localizingand holding the active agent in contact with the skin, nail, hair, clawor hoof, e.g., methylcellulose, sodium carboxymethyl-cellulose, or thelike.

Creams containing the active agent for delivery according to the presentinvention are viscous liquid or semisolid emulsions, either oil-in-wateror water-in-oil. Cream bases are water-washable, and contain an oilphase, an emulsifier and an aqueous phase. The oil phase is generallycomprised of petrolatum or a fatty alcohol, such as cetyl- or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington: TheScience and Practice of Pharmacy, supra, is generally a nonionic,anionic, cationic or amphoteric surfactant.

Gel formulations can also be used in connection with the presentinvention. As will be appreciated by those working in the field oftopical drug formulation, gels are semisolid. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also may be asolvent or solvent blend.

Ointments, which are semisolid preparations, are typically based onpetrolatum or other petroleum derivatives. As will be appreciated by theordinarily skilled artisan, the specific ointment base to be used is onethat provides for optimum delivery for the active agent chosen for agiven formulation, and, preferably, provides for other desiredcharacteristics as well, e.g., emolliency or the like. As with othercarriers or vehicles, an ointment base should be inert, stable,nonirritating and non-sensitizing. As explained in Remington: TheScience and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack PublishingCo., 1995), at pages 1399-1404, ointment bases may be grouped in fourclasses: oleaginous bases; emulsifiable bases; emulsion bases; andwater-soluble bases. Oleaginous ointment bases include, for example,vegetable oils, fats obtained from animals, and semisolid hydrocarbonsobtained from petroleum. Emulsifiable ointment bases, also known asabsorbent ointment bases, contain little or no water and include, forexample, hydroxystearin sulfate, anhydrous lanolin and hydrophilicpetrolatum. Emulsion ointment bases are either water-in-oil (W/O)emulsions or oil-in-water (O/W) emulsions, and include, for example,cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.Preferred water-soluble ointment bases are prepared from polyethyleneglycols of varying molecular weight; again, reference may be had toRemington: The Science and Practice of Pharmacy, supra, for furtherinformation.

Useful formulations of the invention also encompass sprays. Spraysgenerally provide the active agent in an aqueous and/or alcoholicsolution which can be misted onto the skin, nail, hair, claw or hoof fordelivery. Such sprays include those formulated to provide forconcentration of the active agent solution at the site of administrationfollowing delivery, e.g., the spray solution can be primarily composedof alcohol or other like volatile liquid in which the drug or activeagent can be dissolved. Upon delivery to the skin, nail, hair, claw orhoof, the carrier evaporates, leaving concentrated active agent at thesite of administration.

The topical pharmaceutical compositions may also comprise suitable solidor gel phase carriers. Examples of such carriers include but are notlimited to calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols.

The topical pharmaceutical compositions may also comprise a suitableemulsifier which refers to an agent that enhances or facilitates mixingand suspending oil-in-water or water-in-oil. The emulsifying agent usedherein may consist of a single emulsifying agent or may be a nonionic,anionic, cationic or amphoteric surfactant or blend of two or more suchsurfactants; preferred for use herein are nonionic or anionicemulsifiers. Such surface-active agents are described in “McCutcheon'sDetergent and Emulsifiers,” North American Edition, 1980 Annualpublished by the McCutcheon Division, MC Publishing Company, 175 RockRoad, Glen Rock, N.J. 07452, USA.

Preferred for use herein are high molecular weight alcohols such ascetearyl alcohol, cetyl alcohol, stearyl alcohol, emulsifying wax,glyceryl monostearate. Other examples are ethylene glycol distearate,sorbitan tristearate, propylene glycol monostearate, sorbitanmonooleate, sorbitan monostearate (SPAN 60), diethylene glycolmonolaurate, sorbitan monopalmitate, sucrose dioleate, sucrose stearate(CRODESTA F-160), polyoxyethylene lauryl ether (BRIJ 30),polyoxyethylene (2) stearyl ether (BRIJ 72), polyoxyethylene (21)stearyl ether (BRIJ 721), polyoxyethylene monostearate (Myrj 45),polyoxyethylene sorbitan monostearate (TWEEN 60), polyoxyethylenesorbitan monooleate (TWEEN 80), polyoxyethylene sorbitan monolaurate(TWEEN 20) and sodium oleate. Cholesterol and cholesterol derivativesmay also be employed in externally used emulsions and promote w/oemulsions.

Especially suitable nonionic emulsifying agents are those withhydrophile-lipophile balances (HLB) of about 3 to 6 for w/o system and 8to 18 for o/w system as determined by the method described by Paul L.Lindner in “Emulsions and Emulsion”, edited by Kenneth Lissant,published by Dekker, New York, N.Y., 1974, pages 188-190. More preferredfor use herein are one or more nonionic surfactants that produce asystem having HLB of about 8 to about 18.

Examples of such nonionic emulsifiers include but are not limited to“BRIJ 72”, the trade name for a polyoxyethylene (2) stearyl ether havingan HLB of 4.9; “BRIJ 721”, the trade name for a polyoxyethylene (21)stearyl ether having an HLB of 15.5, “Brij 30”, the trade name forpolyoxyethylene lauryl ether having an HLB of 9.7; “Polawax”, the tradename for emulsifying wax having an HLB of 8.0; “Span 60”, the trade namefor sorbitan monostearate having an HLB of 4.7; “Crodesta F-160”, thetrade name for sucrose stearate” having an HLB of 14.5. All of thesematerials are available from Ruger Chemicals Inc.; Croda; ICI Americas,Inc.; Spectrum Chemicals; and BASF. When the topical formulations of thepresent invention contain at least one emulsifying agent, eachemulsifying agent is present in amount from about 0.5 to about 2.5 wt %,preferably 0.5 to 2.0%, more preferably 1.0% or 1.8%. Preferably theemulsifying agent comprises a mixture of steareth 21 (at about 1.8%) andsteareth 2 (at about 1.0%).

The topical pharmaceutical compositions may also comprise suitableemollients. Emollients are materials used for the prevention or reliefof dryness, as well as for the protection of the skin, nail, hair, clawor hoof. Useful emollients include, but are not limited to, cetylalcohol, isopropyl myristate, stearyl alcohol, and the like. A widevariety of suitable emollients are known and can be used herein. Seee.g., Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1,pp. 32-43 (1972), and U.S. Pat. No. 4,919,934, to Deckner et al., issuedApr. 24, 1990, both of which are incorporated herein by reference intheir entirety. These materials are available from Ruger Chemical Co,(Irvington, N.J.).

When the topical formulations of the present invention contain at leastone emollient, each emollient is present in an amount from about 0.1 to15%, preferably 0.1 to about 3.0, more preferably 0.5, 1.0, or 2.5 wt %.Preferably the emollient is a mixture of cetyl alcohol, isopropylmyristate and stearyl alcohol in a 1/5/2 ratio. The emollient may alsobe a mixture of cetyl alcohol and stearyl alcohol in a 1/2 ratio.

The topical pharmaceutical compositions may also comprise suitableantioxidants, substances known to inhibit oxidation. Antioxidantssuitable for use in accordance with the present invention include, butare not limited to, butylated hydroxytoluene, ascorbic acid, sodiumascorbate, calcium ascorbate, ascorbic palmitate, butylatedhydroxyanisole, 2,4,5-trihydroxybutyrophenone,4-hydroxymethyl-2,6-di-tert-butylphenol, erythorbic acid, gum guaiac,propyl gallate, thiodipropionic acid, dilauryl thiodipropionate,tert-butylhydroquinone and tocopherols such as vitamin E, and the like,including pharmaceutically acceptable salts and esters of thesecompounds. Preferably, the antioxidant is butylated hydroxytoluene,butylated hydroxyanisole, propyl gallate, ascorbic acid,pharmaceutically acceptable salts or esters thereof, or mixturesthereof. Most preferably, the antioxidant is butylated hydroxytoluene.These materials are available from Ruger Chemical Co, (Irvington, N.J.).

When the topical formulations of the present invention contain at leastone antioxidant, the total amount of antioxidant present is from about0.001 to 0.5 wt %, preferably 0.05 to about 0.5 wt %, more preferably0.1%.

The topical pharmaceutical compositions may also comprise suitablepreservatives. Preservatives are compounds added to a pharmaceuticalformulation to act as an anti-microbial agent. Among preservatives knownin the art as being effective and acceptable in parenteral formulationsare benzalkonium chloride, benzethonium, chlorohexidine, phenol,m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol,o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal,benzoic acid, and various mixtures thereof. See, e.g., Wallhausser,K.-H., Develop. Biol. Standard, 24:9-28 (1974) (S. Krager, Basel).Preferably, the preservative is selected from methylparaben,propylparaben and mixtures thereof. These materials are available fromInolex Chemical Co (Philadelphia, Pa.) or Spectrum Chemicals.

When the topical formulations of the present invention contain at leastone preservative, the total amount of preservative present is from about0.01 to about 0.5 wt %, preferably from about 0.1 to 0.5%, morepreferably from about 0.03 to about 0.15. Preferably the preservative isa mixture of methylparaben and proplybarben in a 5/1 ratio. When alcoholis used as a preservative, the amount is usually 15 to 20%.

The topical pharmaceutical compositions may also comprise suitablechelating agents to form complexes with metal cations that do not crossa lipid bilayer. Examples of suitable chelating agents include ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and8-Amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraaceticacid, tetrapotassium salt (QUIN-2). Preferably the chelating agents areEDTA and citric acid. These materials are available from SpectrumChemicals.

When the topical formulations of the present invention contain at leastone chelating agent, the total amount of chelating agent present is fromabout 0.005% to 2.0% by weight, preferably from about 0.05% to about 0.5wt %, more preferably about 0.1% by weight.

The topical pharmaceutical compositions may also comprise suitableneutralizing agents used to adjust the pH of the formulation to within apharmaceutically acceptable range. Examples of neutralizing agentsinclude but are not limited to trolamine, tromethamine, sodiumhydroxide, hydrochloric acid, citric acid, and acetic acid. Suchmaterials are available from are available from Spectrum Chemicals(Gardena, Calif.).

When the topical formulations of the present invention contain at leastone neutralizing agent, the total amount of neutralizing agent presentis from about 0.1 wt to about 10 wt %, preferably 0.1 wt % to about 5.0wt %, and more preferably about 1.0 wt %. The neutralizing agent isgenerally added in whatever amount is required to bring the formulationto the desired pH.

The topical pharmaceutical compositions may also comprise suitableviscosity increasing agents. These components are diffusible compoundscapable of increasing the viscosity of a polymer-containing solutionthrough the interaction of the agent with the polymer. CARBOPOL ULTREZ10 may be used as a viscosity-increasing agent. These materials areavailable from Noveon Chemicals, Cleveland, Ohio.

When the topical formulations of the present invention contain at leastone viscosity increasing agent, the total amount of viscosity increasingagent present is from about 0.25% to about 5.0% by weight, preferablyfrom about 0.25% to about 1.0 wt %, and more preferably from about 0.4%to about 0.6% by weight.

The topical pharmaceutical compositions may also comprise suitable nailpenetration enhancers. Examples of nail penetration enhancers includemercaptan compounds, sulfites and bisulfites, keratolytic agents andsurfactants. Nail penetration enhancers suitable for use in theinvention are described in greater detail in Malhotra et al., J. Pharm.Sci., 91:2, 312-323 (2002), which is incorporated herein by reference inits entirety.

The topical pharmaceutical compositions may also comprise one or moresuitable solvents. The ability of any solid substance (solute) todissolve in any liquid substance (solvent) is dependent upon thephysical properties of the solute and the solvent. When solutes andsolvents have similar physical properties the solubility of the solutein the solvent will be the greatest. This gives rise to the traditionalunderstanding that “like dissolves like.” Solvents can be characterizedin one extreme as non-polar, lipophilic oils, while in the other extremeas polar hydrophilic solvents. Oily solvents dissolve other non-polarsubstances by Van der Wals interactions while water and otherhydrophilic solvents dissolve polar substances by ionic, dipole, orhydrogen bonding interactions. All solvents can be listed along acontinuum from the least polar, i.e. hydrocarbons such as decane, to themost polar solvent being water. A solute will have its greatestsolubility in solvents having equivalent polarity. Thus, for drugshaving minimal solubility in water, less polar solvents will provideimproved solubility with the solvent having polarity nearly equivalentto the solute providing maximum solubility. Most drugs have intermediatepolarity, and thus experience maximum solubility in solvents such aspropylene glycol or ethanol, which are significantly less polar thanwater. If the drug has greater solubility in propylene glycol (forexample 8% (w/w)) than in water (for example 0.1% (w/w)), then additionof water to propylene glycol should decrease the maximum amount of drugsolubility for the solvent mixture compared with pure propylene glycol.Addition of a poor solvent to an excellent solvent will decrease themaximum solubility for the blend compared with the maximum solubility inthe excellent solvent.

When compounds are incorporated into topical formulations theconcentration of active ingredient in the formulation may be limited bythe solubility of the active ingredient in the chosen solvent and/orcarrier. Non-lipophilic drugs typically display very low solubility inpharmaceutically acceptable solvents and/or carriers. For example, thesolubility of some compounds in the invention in water is less than0.00025% wt/wt. The solubility of the same compounds in the inventioncan be less than about 2% wt/wt in either propylene glycol or isopropylmyristate. In one embodiment of the present invention, diethylene glycolmonoethyl ether (DGME) is the solvent used to dissolve the compounds ofthe invention. The compounds in the invention useful in the presentformulation are believed to have a solubility of from about 10% wt/wt toabout 25% wt/wt in DGME. In another embodiment a DGME water cosolventsystem is used to dissolve the compounds of the invention. The solventcapacity of DGME drops when water is added; however, the DGME/watercosolvent system can be designed to maintain the desired concentrationof from about 0.1% to about 5% wt/wt active ingredient. Preferably theactive ingredient is present from about 0.5% to about 3% wt/wt, and morepreferably at about 1% wt/wt, in the as-applied topical formulations.Because DGME is less volatile than water, as the topical formulationevaporates upon application, the active agent becomes more soluble inthe cream formulation. This increased solubility reduces the likelihoodof reduced bioavailability caused by the drug precipitating on thesurface of the skin, nail, hair, claw or hoof.

Liquid forms, such as lotions suitable for topical administration orsuitable for cosmetic application, may include a suitable aqueous ornonaqueous vehicle with buffers, suspending and dispensing agents,thickeners, penetration enhancers, and the like. Solid forms such ascreams or pastes or the like may include, for example, any of thefollowing ingredients, water, oil, alcohol or grease as a substrate withsurfactant, polymers such as polyethylene glycol, thickeners, solids andthe like. Liquid or solid formulations may include enhanced deliverytechnologies such as liposomes, microsomes, microsponges and the like.

Additionally, the compounds can be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.

Topical treatment regimens according to the practice of this inventioncomprise applying the composition directly to the skin, nail, hair, clawor hoof at the application site, from one to several times daily.

Formulations of the present invention can be used to treat, ameliorateor prevent conditions or symptoms associated with bacterial infections,acne, inflammation and the like.

In an exemplary embodiment, the pharmaceutical formulation includes asimple solution. In an exemplary embodiment, the simple solutionincludes an alcohol. In an exemplary embodiment, the simple solutionincludes alcohol and water. In an exemplary embodiment, the alcohol isethanol, ethylene glycol, propanol, polypropylene glycol, isopropanol orbutanol. In another exemplary embodiment, the simple solution is amember selected from about 10% polypropylene glycol and about 90%ethanol; about 20% polypropylene glycol and about 80% ethanol; about 30%polypropylene glycol and about 70% ethanol; about 40% polypropyleneglycol and about 60% ethanol; about 50% polypropylene glycol and about50% ethanol; about 60% polypropylene glycol and about 40% ethanol; about70% polypropylene glycol and about 30% ethanol; about 80% polypropyleneglycol and about 20% ethanol; about 90% polypropylene glycol and about10% ethanol.

In an exemplary embodiment, the pharmaceutical formulation is a lacquer.Please see Remington's, supra, for more information on the production oflacquers.

In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 0.5% toabout 15%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 0.1% toabout 12.5%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 1% to about10%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 1% to about5%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 0.5% toabout 5%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 0.5% toabout 7.5%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 5% to about7.5%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 2% to about8%. In an exemplary embodiment, the compound is present in saidpharmaceutical formulation in a concentration of from about 4% to about9%.

VII. b) Additional Active Agents

The following are examples of the cosmetic and pharmaceutical agentsthat can be added to the topical pharmaceutical formulations of thepresent invention. The following agents are known compounds and arereadily available commercially.

Anti-inflammatory agents include, but are not limited to, bisabolol,mentholatum, dapsone, aloe, hydrocortisone, and the like.

Vitamins include, but are not limited to, Vitamin B, Vitamin E, VitaminA, Vitamin D, and the like and vitamin derivatives such as tazarotene,calcipotriene, tretinoin, adapalene and the like.

Anti-aging agents include, but are not limited to, niacinamide, retinoland retinoid derivatives, AHA, Ascorbic acid, lipoic acid, coenzyme Q10, beta hydroxy acids, salicylic acid, copper binding peptides,dimethylaminoethyl (DAEA), and the like.

Sunscreens and or sunburn relief agents include, but are not limited to,PABA, jojoba, aloe, padimate-O, methoxycinnamates, proxamine HCl,lidocaine and the like. Sunless tanning agents include, but are notlimited to, dihydroxyacetone (DHA).

Psoriasis-treating agents and/or acne-treating agents include, but arenot limited to, salicylic acid, benzoyl peroxide, coal tar, seleniumsulfide, zinc oxide, pyrithione (zinc and/or sodium), tazarotene,calcipotriene, tretinoin, adapalene and the like.

Agents that are effective to control or modify keratinization, includingwithout limitation: tretinoin, tazarotene, and adapalene.

The compositions comprising an compound/active agent of the invention,and optionally at least one of these additional agents, are to beadministered topically. In a primary application, this leads to thecompounds of the invention and any other active agent working upon andtreating the skin, nail, hair, claw or hoof. Alternatively, any one ofthe topically applied active agents may also be delivered systemicallyby transdermal routes.

In such compositions an additional cosmetically or pharmaceuticallyeffective agent, such as an anti-inflammatory agent, vitamin, anti-agingagent, sunscreen, and/or acne-treating agent, for example, is usually aminor component (from about 0.001% to about 20% by weight or preferablyfrom about 0.01% to about 10% by weight) with the remainder beingvarious vehicles or carriers and processing aids helpful for forming thedesired dosing form.

VII. c) Testing

Preferred compounds for use in the present topical formulations willhave certain pharmacological properties. Such properties include, butare not limited to, low toxicity, low serum protein binding anddesirable in vitro and in vivo half-lives. Assays may be used to predictthese desirable pharmacological properties. Assays used to predictbioavailability include transport across human intestinal cellmonolayers, including Caco-2 cell monolayers. Serum protein binding maybe predicted from albumin binding assays. Such assays are described in areview by Oravcova et al. (1996, J. Chromat. B677: 1-27). Compoundhalf-life is inversely proportional to the frequency of dosage of acompound. In vitro half-lives of compounds may be predicted from assaysof microsomal half-life as described by Kuhnz and Gleschen (DrugMetabolism and Disposition, (1998) volume 26, pages 1120-1127).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD₅₀and ED₅₀. Compounds that exhibit high therapeutic indices are preferred.The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1, p. 1).

VII. d) Administration

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays, as disclosed herein. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the EC₅₀ (effective dose for 50% increase) as determinedin cell culture, i.e., the concentration of the test compound whichachieves a half-maximal inhibition of bacterial cell growth. Suchinformation can be used to more accurately determine useful doses inhumans.

In general, the compounds prepared by the methods, and from theintermediates, described herein will be administered in atherapeutically or cosmetically effective amount by any of the acceptedmodes of administration for agents that serve similar utilities. It willbe understood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination, the severity of the particular diseaseundergoing therapy and the judgment of the prescribing physician. Thedrug can be administered from once or twice a day, or up to 3 or 4 timesa day.

Dosage amount and interval can be adjusted individually to provideplasma levels of the active moiety that are sufficient to maintainbacterial cell growth inhibitory effects. Usual patient dosages forsystemic administration range from 0.1 to 1000 mg/day, preferably, 1-500mg/day, more preferably 10-200 mg/day, even more preferably 100-200mg/day. Stated in terms of patient body surface areas, usual dosagesrange from 50-91 mg/m²/day.

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-10 wt %of the drug based on the total formulation, with the balance being oneor more suitable pharmaceutical excipients. Preferably, the compound ispresent at a level of about 0.1-3.0 wt %, more preferably, about 1.0 wt%.

The invention is further illustrated by the Examples that follow. TheExamples are not intended to define or limit the scope of the invention.

EXAMPLES

Proton NMR are recorded on Varian AS 300 spectrometer and chemicalshifts are reported as δ (ppm) down field from tetramethylsilane. Massspectra are determined on Micromass Quattro II.

Example 1 Preparation of 3 from 1 1.1 Reduction of Carboxylic Acid

To a solution of 1 (23.3 mmol) in anhydrous THF (70 mL) under nitrogenwas added dropwise a BH₃THF solution (1.0 M, 55 mL, 55 mmol) at 0° C.and the reaction mixture was stirred overnight at room temperature. Thenthe mixture was cooled again with ice bath and MeOH (20 mL) was addeddropwise to decompose excess BH₃. The resulting mixture was stirreduntil no bubble was released and then 10% NaOH (10 mL) was added. Themixture was concentrated and the residue was mixed with water (200 mL)and extracted with EtOAc. The residue from rotary evaporation waspurified by flash column chromatography over silica gel to give 20.7mmol of 3.

1.2 Results

Exemplary compounds of structure 3 prepared by the method above areprovided below.

1.2.a 2-Bromo-5-chlorobenzyl Alcohol

¹H NMR (300 MHz, DMSO-d₆): δ 7.57 (d, J=8.7 Hz, 1H), 7.50-7.49 (m, 1H),7.28-7.24 (m, 1H), 5.59 (t, J=6.0 Hz, 1H) and 4.46 (d, J=6.0 Hz, 2H)ppm.

1.2.b 2-Bromo-5-methoxybenzyl Alcohol

¹H NMR (300 MHz, DMSO-d₆): δ 7.42 (d, J=8.7 Hz, 1H), 7.09 (d, J=2.4 Hz,1H), 6.77 (dd, J=3 Hz, J₂=3 Hz, 1H), 5.43 (t, J=5.7 Hz, 1H), 4.44 (d,J=5.1 Hz, 2H), 3.76 (s, 3H).

Example 2 Preparation of 3 from 2 2.1. Reduction of Aldehyde

To a solution of 2 (Z═H, 10.7 mmol) in methanol (30 mL) was added sodiumborohydride (5.40 mol), and the mixture was stirred at room temperaturefor 1 h. Water was added, and the mixture was extracted with ethylacetate. The organic layer was washed with brine and dried on anhydroussodium sulfate. The solvent was removed under reduced pressure to afford9.9 mmol of 3.

2.2 Results

Exemplary compounds of structure 3 prepared by the method above areprovided below.

2.2.a 2-Bromo-5-(4-cyanophenoxy)benzyl Alcohol

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 2.00 (br s, 1H), 4.75 (s, 2H), 6.88 (dd,J=8.5, 2.9 Hz, 1H), 7.02 (d, J=8.8 Hz, 1H), 7.26 (d, J=2.6 Hz, 1H), 7.56(d, J=8.5 Hz, 1H), 7.62 (d, J=8.8 Hz, 2H).

2.2.b 2-Bromo-4-(4-cyanophenoxy)benzyl Alcohol

¹H NMR (300 MHz, DMSO-d₆): δ 7.83 (d, 2H), 7.58 (d, 1H), 7.39 (d, 1H),7.18 (dd, 1H), 7.11 (d, 2H), 5.48 (t, 1H) and 4.50 (d, 2H) ppm.

2.2.c 5-(4-Cyanophenoxy)-1-Indanol

M.p. 50-53° C. MS (ESI+): m/z=252 (M+1). HPLC: 99.7% purity at 254 nmand 99.0% at 220 nm. ¹H NMR (300 MHz, DMSO-d₆): δ 7.80 (d, 2H), 7.37 (d,1H), 7.04 (d, 2H), 6.98-6.93 (m, 2H), 5.27 (d, 1H), 5.03 (q, 1H),2.95-2.85 (m, 1H), 2.75-2.64 (m, 1H), 2.39-2.29 (m, 1H) and 1.85-1.74(m, 1H) ppm.

2.2.d 2-Bromo-5-(tert-butyldimethylsiloxy)benzyl Alcohol

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 0.20 (s, 6H), 0.98 (s, 9H), 4.67 (br s,1H), 6.65 (dd, J=8.2, 2.6 Hz, 1H), 6.98 (d, J=2.9 Hz, 1H), 7.36 (d,J=8.8 Hz, 1H).

Additional examples of compounds which can be produced by this methodinclude 2-bromo-4-(3-cyanophenoxy)benzyl alcohol;2-bromo-4-(4-chlorophenoxy)benzyl alcohol; 2-bromo-4-phenoxybenzylalcohol; 2-bromo-5-(3,4-dicyanophenoxy)benzyl alcohol;2-(2-bromo-5-fluorophenyl)ethyl alcohol; 2-bromo-5-fluorobenzyl alcohol;and 1-bromo-2-naphthalenemethanol.

Example 3 Preparation of 4 from 3 3.1 Protective Alkylation

Compound 3 (20.7 mmol) was dissolved in CH₂Cl₂ (150 mL) and cooled to 0°C. with ice bath. To this solution under nitrogen were added in sequenceN,N-di-isopropyl ethyl amine (5.4 mL, 31.02 mmol, 1.5 eq) andchloromethyl methyl ether (2 mL, 25.85 mmol, 1.25 eq). The reactionmixture was stirred overnight at room temperature and washed withNaHCO₃-saturated water and then NaCl-saturated water. The residue afterrotary evaporation was purified by flash column chromatography oversilica gel to give 17.6 mmol of 4.

3.2 Results

Exemplary compounds of structure 4 prepared by the method above areprovided below.

3.2.a 2-Bromo-5-chloro-1-(methoxymethoxymethyl)benzene

¹H NMR (300 MHz, DMSO-d₆): δ 7.63 (d, J=8.7 Hz, 1H), 7.50 (dd, J=2.4 &0.6 Hz, 1H), 7.32 (dd, J=8.4 & 2.4 Hz, 1H), 4.71 (s, 2H), 4.53 (s, 2H)and 3.30 (s, 3H) ppm.

3.2.b 2-Bromo-5-fluoro-1-[1-(methoxymethoxy)ethyl]benzene

¹H-NMR (300.058 MHz, CDCl₃) δ ppm 1.43 (d, J=6.5 Hz, 3H), 3.38 (s, 3H),4.55 (d, J=6.5 Hz, 1H), 4.63 (d, J=6.5 Hz, 1H), 5.07 (q, J=6.5 Hz, 1H),6.85 (m, 1H), 7.25 (dd, J=9.7, 2.6 Hz, 1H), 7.46 (dd, J=8.8, 5.3 Hz,1H).

3.2.c 2-Bromo-5-fluoro-1-[2-(methoxymethoxy)ethyl]benzene

¹H-NMR (300.058 MHz, CDCl₃) δ ppm 3.04 (t, J=6.7 Hz, 2H), 3.31 (s, 3H),3.77 (t, J=6.7 Hz, 2H), 4.62 (s, 2H), 6.82 (td, J=8.2, 3.2 Hz, 1H), 7.04(dd, J=9.4, 2.9 Hz, 1H), 7.48 (dd, J=8.8, 5.3 Hz, 1H).

3.2.d 2-Bromo-4,5-difluoro-1-(methoxymethoxymethyl)benzene

¹H-NMR (300.058 MHz, CDCl₃) δ ppm 3.42 (s, 3H), 4.57 (d, J=1.2 Hz, 2H),4.76 (s, 2H), 7.3-7.5 (m, 2H).

3.2.e 2-Bromo-5-cyano-1-(methoxymethoxymethyl)benzene

¹H-NMR (300.058 MHz, CDCl₃) δ ppm 3.43 (s, 3H), 4.65 (s, 2H), 4.80 (s,2H), 7.43 (dd, J=8.2, 4.1 Hz, 1H), 7.66 (d, J=8.2 Hz, 1H), 7.82 (d,J=4.1 Hz, 1H).

3.2.f 2-Bromo-5-methoxy(methoxymethoxymethyl)benzene

¹H NMR (300 MHz, DMSO-d₆): δ 7.48 (dd, J₁=1.2 Hz, J₂=1.2 Hz, 1H), 7.05(d, J=2.7 Hz, 1H), 6.83 (dd, J₁=3 Hz, J₂=3 Hz, 1H), 4.69 (d, J=1.2 Hz,2H), 4.5 (s, 2H), 3.74 (d, J=1.5 Hz, 3H), 3.32 (d, J=2.1 Hz, 3H) ppm.

3.2.g 1-Benzyl-1-(2-bromophenyl)-1-(methoxymethoxy)ethane

¹H NMR (300 MHz, DMSO-d₆): δ 7.70-7.67 (m, 1H), 7.25-7.09 (m, 6H),6.96-6.93 (m, 2H), 4.61 (d, 1H), 4.48 (d, 1H), 3.36-3.26 (m, 2H), 3.22(s, 3H) and 1.63 (s, 3H) ppm.

3.2.h 2-Bromo-6-fluoro-1-(methoxmethoxymethoxymethyl)benzene

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 3.43 (s, 3H), 4.74 (s, 2H), 4.76 (d,J=2.1 Hz, 2H), 7.05 (t, J=9.1 Hz, 1H), 7.18 (td, J=8.2, 5.9 Hz, 1H),7.40 (d, J=8.2 Hz, 1H).

3.2.i 2-Bromo-4-(4-cyanophenoxy)-1-(methoxymethoxymethyl)benzene

¹H NMR (300 MHz, DMSO-d₆): δ 7.84 (d, 2H), 7.56 (d, 1H), 7.44 (d, 1H),7.19-7.12 (m, 3H), 4.69 (s, 2H), 4.56 (s, 2H) and 3.31 (s, 3H) ppm.

3.2.j2-Bromo-5-(tert-butyldimethylsiloxy)-1-(methoxymethoxymethyl)benzene

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 0.19 (s, 6H), 0.98 (s, 9H), 3.43 (s,3H), 4.59 (s, 2H), 4.75 (s, 2H), 6.64 (dd, J=8.5, 2.9 Hz, 1H), 6.98 (d,J=2.9 Hz, 1H), 7.36 (d, J=8.5 Hz, 1H).

3.2.k 2-Bromo-5-(2-cyanophenoxy)-1-(methoxymethoxymethyl)benzene

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 3.41 (s, 3H), 4.64 (s, 2H), 4.76 (s,2H), 6.8-6.9 (m, 2H), 7.16 (td, J=7.6, 0.9 Hz, 1H), 7.28 (d, J=2.9 Hz,1H), 7.49 (ddd, J=8.8, 7.6, 1.8 Hz, 1H), 7.56 (d, J=8.5 Hz, 1H), 7.67(dd, J=7.9, 1.8 Hz, 1H).

3.2.1 2-Bromo-5-phenoxy-1-(methoxymethoxymethyl)benzene

¹H-NMR (300 MHz, CDCl₃) δ (ppm) 3.40 (s, 3H), 4.62 (s, 2H), 4.74 (s,2H), 6.80 (dd, J=8.8, 2.9 hz, 1H), 7.01 (d, J=8.5 Hz, 2H), 7.12 (t,J=7.9 Hz, 1H), 7.19 (d, J=2.9 hz, 1H), 7.35 (t, J=7.6 Hz, 2H), 7.48 (d,J=8.5 Hz, 1H).

Additional examples of compounds which can be produced by this methodinclude 2-bromo-1-(methoxymethoxymethyl)benzene;2-bromo-5-methyl-1-(methoxymethoxymethyl)benzene;2-bromo-5-(methoxymethoxymethyl)-1-(methoxymethoxymethyl)benzene;2-bromo-5-fluoro-1-(methoxymethoxymethyl)benzene;1-bromo-2-(methoxymethoxymethyl)naphthalene;2-bromo-4-fluoro-1-(methoxymethoxymethyl)benzene;2-phenyl-1-(2-bromophenyl)-1-(methoxymethoxy)ethane;2-bromo-5-(4-cyanophenoxy)-1-(methoxymethoxy methyl)benzene;2-bromo-4-(3-cyanophenoxy)-1-(methoxymethoxymethyl)benzene;2-bromo-4-(4-chlorophenoxy)-1-(methoxymethoxymethyl)benzene;2-bromo-4-phenoxy-1-(methoxymethoxymethyl)benzene;2-bromo-5-(3,4-dicyanophenoxy)-1-(methoxymethoxymethyl)benzene.

Example 4

Preparation of I from 4 Via 5

4.1 Metallation and Boronylation

To a solution of 4 (17.3 mmol) in anhydrous THF (80 mL) at −78° C. undernitrogen was added dropwise tert-BuLi or n-BuLi (11.7 mL) and thesolution became brown colored. Then, B(OMe)₃ (1.93 mL, 17.3 mmol) wasinjected in one portion and the cooling bath was removed. The mixturewas warmed gradually with stirring for 30 min and then stirred with awater bath for 2 h. After addition of 6N HCl (6 mL), the mixture wasstirred overnight at room temperature and about 50% hydrolysis hashappened as shown by TLC analysis. The solution was rotary evaporatedand the residue was dissolved in MeOH (50 mL) and 6N HCl (4 mL). Thesolution was refluxed for 1 h and the hydrolysis was completed asindicated by TLC analysis. Rotary evaporation gave a residue which wasdissolved in EtOAc, washed with water, dried and then evaporated. Thecrude product was purified by flash column chromatography over silicagel to provide a solid with 80% purity. The solid was further purifiedby washing with hexane to afford 7.2 mmol of I.

4.2 Results

Analytical data for exemplary compounds of structure I are providedbelow.

4.2.a 5-Chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole5-chlorobenzo[c][1,2]oxaborol-1(3H)-ol (C1)

M.p. 142-150° C. MS (ESI): m/z=169 (M+1, positive) and 167 (M−1,negative). HPLC (220 nm): 99% purity. ¹H NMR (300 MHz, DMSO-d₆): δ 9.30(s, 1H), 7.71 (d, J=7.8 Hz, 1H), 7.49 (s, 1H), 7.38 (d, J=7.8 Hz, 1H)and 4.96 (s, 2H) ppm.

4.2.b 1,3-Dihydro-1-hydroxy-2,1-benzoxaborolebenzo[c][1,2]oxaborol-1(3H)-ol (C2)

M.p. 83-86° C. MS (ESI): m/z=135 (M+1, positive) and 133 (M−1,negative). HPLC (220 nm): 95.4% purity. ¹H NMR (300 MHz, DMSO-d₆): δ9.14 (s, 1H), 7.71 (d, J=7.2 Hz, 1H), 7.45 (t, J=7.5 Hz, 1H), 7.38 (d,J=7.5 Hz, 1H), 7.32 (t, J=7.1 Hz, 1H) and 4.97 (s, 2H) ppm.

4.2.c 5-chloro-3-methylbenzo[c][1,2]oxaborol-1(3H)-ol (C3)

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 1.37 (d, J=6.4 Hz, 3H), 5.17 (q, J=6.4Hz, 1H), 7.14 (m, 1H), 7.25 (dd, J=9.7, 2.3 Hz, 1H), 7.70 (dd, J=8.2,5.9 Hz, 1H), 9.14 (s, 1H).

4.2.d 6-Fluoro-1-hydroxy-1,2,3,4-tetrahydro-2,1-benzoxaborine6-fluoro-3,4-dihydrobenzo[c][1,2]oxaborinin-1-ol (C4)

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 2.86 (t, J=5.9 Hz, 2H), 4.04 (t, J=5.9Hz, 2H), 7.0-7.1 (m, 2H), 7.69 (dd, J=8.2, 7.2 Hz, 1H), 8.47 (s, 1H).

4.2.e 5,6-Difluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole5,6-difluorobenzo[c][1,2]oxaborol-1(3H)-ol (C5)

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 4.94 (s, 2H), 7.50 (dd, J=10.7, 6.8 Hz,1H), 7.62 (dd, J=9.7, 8.2 Hz, 1H), 9.34 (s, 1H).

4.2.f 5-Cyano-1,3-dihydro-1-hydroxy-2,1-benzoxaborole1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbonitrile (C6)

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 5.03 (s, 2H), 7.76 (d, J=8.2 Hz, 1H),7.89 (d, J=8.2 Hz, 1H), 7.90 (s, 1H), 9.53 (s, 1H).

4.2.g 1,3-Dihydro-1-hydroxy-5-methoxy-2,1-benzoxaborole5-methoxybenzo[c][1,2]oxaborol-1(3H)-ol (C7)

M.p. 102-104° C. MS ESI: m/z=165.3 (M+1) and 162.9 (M−1). ¹H NMR (300MHz, DMSO-d₆): δ 8.95 (s, 1H), 7.60 (d, J=8.1 Hz, 1H), 6.94 (s, 1H),6.88 (d, J=8.1 Hz, 1H), 4.91 (s, 2H), 3.77 (s, 3H) ppm.

4.2.h 1,3-Dihydro-1-hydroxy-5-methyl-2,1-benzoxaborole5-methylbenzo[c][1,2]oxaborol-1(3H)-ol (C8)

M.p. 124-128° C. MS ESI: m/z=148.9 (M+1) and 146.9 (M−1). ¹H NMR (300MHz, DMSO-d₆): δ 9.05 (s, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.18 (s, 1H),7.13 (d, J=7.2 Hz, 2H), 4.91 (s, 2H), 2.33 (s, 3H) ppm.

4.2.i 1,3-Dihydro-1-hydroxy-5-hydroxymethyl-2,1-benzoxaborole5-(hydroxymethyl)benzo[c][1,2]oxaborol-1(3H)-ol (C9)

MS: m/z=163 (M−1, ESI−). ¹H NMR (300 MHz, DMSO-d₆): δ 9.08 (s, 1H), 7.64(d, 1H), 7.33 (s, 1H), 7.27 (d, 1H), 5.23 (t, 1H), 4.96 (s, 2H), 4.53(d, 2H) ppm.

4.2.j 1,3-Dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C1O)

M.p. 110-114° C. MS ESI: m/z=150.9 (M−1). ¹H NMR (300 MHz, DMSO-d₆): δ9.20 (s, 1H), 7.73 (dd, J₁=6 Hz, J₂=6 Hz, 1H), 7.21 (m, 1H), 7.14 (m,1H), 4.95 (s, 2H) ppm.

4.2.k 1,3-Dihydro-2-oxa-1-cyclopenta[{acute over (α)}]naphthalenenaphtho[1,2-c][1,2]oxaborol-1(3H)-ol (C11)

M.P. 139-143° C. MS ESI: m/z=184.9 (M+1). ¹H NMR (300 MHz, DMSO-d₆): δ9.21 (s, 1H), 8.28 (dd, J₁=6.9 Hz, J₂=0.6 Hz, 1H), 7.99 (d, J=8.1 Hz,1H), 7.95 (d, J=7.5 Hz, 1H), 7.59-7.47 (m, 3H), 5.09 (s, 2H) ppm.

4.2.m 1,3-Dihydro-6-fluoro-1-hydroxy-2,1-benzoxaborole6-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C13)

M.p. 110-117.5° C. MS (ESI): m/z=151 (M−1, negative). HPLC (220 nm):100% purity. ¹H NMR (300 MHz, DMSO-d₆): δ 9.29 (s, 1H), 7.46-7.41 (m,2H), 7.29 (td, 1H) and 4.95 (s, 2H) ppm.

4.2.n 3-Benzyl-1,3-dihydro-1-hydroxy-3-methyl-2,1-benzoxaborole3-benzyl-3-methylbenzo[c][1,2]oxaborol-1(3H)-ol (C14)

MS (ESI): m/z=239 (M+1, positive). HPLC: 99.5% purity at 220 nm and95.9% at 254 nm. ¹H NMR (300 MHz, DMSO-d₆): δ 8.89 (s, 1H), 7.49-7.40(m, 3H), 7.25-7.19 (m, 1H), 7.09-7.05 (m, 3H), 6.96-6.94 (m, 2H), 3.10(d, 1H), 3.00 (d, 1H) and 1.44 (s, 3H) ppm.

4.2.0 3-Benzyl-1,3-dihydro-1-hydroxy-2,1-benzoxaborole3-benzylbenzo[c][1,2]oxaborol-1(3H)-ol (C15)

MS (ESI+): m/z=225 (M+1). HPLC: 93.4% purity at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.08 (s, 1H), 7.63 (dd, 1H), 7.43 (t, 1H), 7.35-7.14 (m,7H), 5.38 (dd, 1H), 3.21 (dd, 1H) and 2.77 (dd, 1H) ppm.

4.2.p 1,3-Dihydro-4-fluoro-1-hydroxy-2,1-benzoxaborole4-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C16)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 5.06 (s, 2H), 7.26 (ddd, J=9.7, 7.9,0.6 Hz, 1H), 7.40 (td, J=8.2, 4.7 Hz, 1H), 7.55 (d, J=7.0 Hz, 1H), 9.41(s, 1H).

4.2.q 5-(4-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzonitrile (C17)

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 4.95 (s, 2H), 7.08 (dd, J=7.9, 2.1 Hz,1H), 7.14 (d, J=8.8 Hz, 1H), 7.15 (d, J=2.1 Hz, 1H), 7.78 (d, J=7.9 Hz,1H), 7.85 (d, J=9.1 Hz, 2H), 9.22 (s, 1H).

4.2.r 6-(4-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)benzonitrile (C18)

M.p. 148-151° C. MS: m/z=252 (M+1) (ESI+) and m/z=250 (M−1) (ESI−).HPLC: 100% purity at 254 nm and 98.7% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.26 (s, 1H), 7.82 (d, 2H), 7.50 (d, 1H), 7.39 (d, 1H), 7.26(dd, 1H), 7.08 (d, 2H) and 4.99 (s, 2H) ppm

4.2.s 6-(3-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole3-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yloxy)benzonitrile (C19)

M.p. 146-149° C. MS: m/z=252 (M+1) (ESI+) and m/z=250 (M−1) (ESI−).HPLC: 100% purity at 254 nm and 97.9% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.21 (s, 1H), 7.60-7.54 (m, 2H), 7.50-7.45 (m, 2H),7.34-7.30 (m, 2H), 7.23 (dd, 1H) and 4.98 (s, 2H) ppm.

4.2.t 6-(4-Chlorophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-chlorophenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C20)

M.p. 119-130° C. MS: m/z=261 (M+1) (ESI+) and m/z=259 (M−1) (ESI−).HPLC: 100% purity at 254 nm and 98.9% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.18 (s, 1H), 7.45-7.41 (m, 3H), 7.29 (d, 1H), 7.19 (dd,1H), 7.01 (d, 2H) and 4.96 (s, 2H) ppm.

4.2.u 6-Phenoxy-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-phenoxybenzo[c][1,2]oxaborol-1(3H)-ol (C21)

M.p. 95-99° C. MS: m/z=227 (M+1) (ESI+) and m/z=225 (M−1) (ESI−). HPLC:100% purity at 254 nm and 98.4% at 220 nm. ¹H NMR (300 MHz, DMSO-d₆): δ9.17 (s, 1H), 7.43-7.35 (m, 3H), 7.28 (s, 1H), 7.19-7.09 (m, 2H), 6.99(d, 2H) and 4.96 (s, 2H) ppm.

4.2.v 5-(4-Cyanobenzyloxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole4-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)methyl)benzonitrile(C22)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.90 (s, 2H), 5.25 (s, 2H), 6.98 (dd,J=7.9, 2.1 Hz, 1H), 7.03 (d, J=1.8 Hz, 1H), 7.62 (d, J=7.9 Hz, 1H), 7.64(d, J=8.5 Hz, 2H), 7.86 (d, J=8.5 Hz, 1H), 9.01 (s, 1H).

4.2.w 5-(2-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzonitrile (C23)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.95 (s, 2H), 7.0-7.2 (m, 3H), 7.32(td, J=7.6, 1.2 Hz, 1H), 7.68 (ddd, J=9.1, 7.6, 1.8 Hz, 1H), 7.77 (d,J=7.9 Hz, 1H), 7.91 (dd, J=7.9, 1.8 Hz, 1H).

4.2.x 5-Phenoxy-1,3-dihydro-1-hydroxy-2,1-benzoxaborole5-phenoxybenzo[c][1,2]oxaborol-1(3H)-ol (C24)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.91 (s, 2H), 6.94 (s, 1H), 6.96 (d,J=8.8 Hz, 1H), 7.05 (d, J=7.6 Hz, 2H), 7.17 (t, J=7.3 Hz, 1H), 7.41 (t,J=7.3 Hz, 2H), 7.70 (d, J=8.5 Hz, 1H), 9.11 (s, 1H).

4.2.y5-[4-(N,N-Diethylcarbamoyl)phenoxy]-1,3-dihydro-1-hydroxy-2,1-benzoxaboroleN,N-diethyl-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzamide(C25)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.08 (br s, 6H), 3.1-3.5 (m, 4H), 4.93(s, 2H), 7.0-7.1 (m, 4H), 7.37 (d, J=8.5 Hz, 2H), 7.73 (d, J=7.9 Hz,1H), 9.15 (s, 1H).

4.2.z1,3-Dihydro-1-hydroxy-5-[4-(morpholinocarbonyl)phenoxy]-2,1-benzoxaborole(4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)phenyl)(morpholino)methanone(C26)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 3.3-3.7 (m, 8H), 4.93 (s, 2H), 7.0-7.1(m, 4H), 7.44 (d, J=8.8 Hz, 2H), 7.73 (d, J=7.9 Hz, 1H), 9.16 (s, 1H).

4.2.aa 5-(3,4-Dicyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)phthalonitrile(C27)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.97 (s, 2H), 7.13 (dd, J=7.9, 2.1 Hz,1H), 7.21 (d, J=1.5 Hz, 1H), 7.43 (dd, J=8.8, 2.6 Hz, 1H), 7.81 (d,J=7.9 Hz, 1H), 7.82 (d, J=2.6 Hz, 1H), 8.11 (d, J=8.5 Hz, 1H), 9.26 (s,1H).

4.2.ab 6-Phenylthio-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(phenylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C28)

M.p. 121-124° C. MS: m/z=243 (M+1) (ESI+) and m/z=241 (M−1) (ESI−).HPLC: 99.6% purity at 254 nm and 99.6% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.25 (s, 1H), 7.72 (dd, 1H), 7.48 (dd, 1H), 7.43 (dd, 1H),7.37-7.31 (m, 2H), 7.29-7.23 (m, 3H), and 4.98 (s, 2H) ppm.

4.2.ac6-(4-trifluoromethoxyphenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-(trifluoromethoxy)phenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C29)

M.p. 97-101° C. MS: m/z=311 (M+1) (ESI+) and m/z=309 (M−1) (ESI−). HPLC:100% purity at 254 nm and 100% at 220 nm. ¹H NMR (300 MHz, DMSO-d₆): δ9.20 (s, 1H), 7.45 (d, 1H), 7.37 (d, 2H), 7.33 (d, 1H), 7.21 (dd, 1H),7.08 (d, 2H), and 4.97 (s, 2H) ppm.

4.2.ad5-(N-Methyl-N-phenylsulfonylamino)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroleN-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)-N-methylbenzenesulfonamide(C30)

M.p. 85-95° C. MS: m/z=304 (M+1) (ESI+) and m/z=302 (M−1) (ESI−). HPLC:96.6% purity at 254 nm and 89.8% at 220 nm. ¹H NMR (300 MHz, DMSO-d₆): δ9.23 (s, 1H), 7.72-7.63 (m, 2H), 7.56 (t, 2H), 7.50 (d, 2H), 7.16 (s,1H), 7.03 (d, 1H), 4.91 (s, 2H) and 3.14 (s, 3H) ppm.

4.2.ae 6-(4-Methoxyphenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-methoxyphenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C31)

M.p. 126-129° C. MS: m/z=257 (M+1) (ESI+) and m/z=255 (M−1) (ESI−).HPLC: 98.4% purity at 254 nm and 98.4% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.14 (s, 1H), 7.36 (d, 1H), 7.19 (s, 1H), 7.12 (d, 1H), 6.98(d, 2H), 6.95 (d, 2H), 4.93 (s, 2H) and 3.73 (s, 3H) ppm.

4.2.af 6-(4-Methoxyphenylthio)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-methoxyphenylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C32)

M.p. 95-100° C. MS: m/z=272 (M+), 273 (M+1) (ESI+) and m/z=271 (M−1)(ESI−). HPLC: 100% purity at 254 nm and 99.2% at 220 nm. ¹H NMR (300MHz, DMSO-d₆): δ 9.20 (s, 1H), 7.51 (d, 1H), 7.39-7.28 (m, 4H), 6.98 (d,2H), 4.93 (s, 2H) and 3.76 (s, 3H) ppm.

4.2.ag6-(4-Methoxyphenylsulfonyl)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-methoxyphenylsulfonyl)benzo[c][1,2]oxaborol-1(3H)-ol (C33)

M.p. 180-192° C. MS: m/z=305 (M+1) (ESI+) and m/z=303 (M−1) (ESI−).HPLC: 96.8% purity at 254 nm and 95.5% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.46 (s, 1H), 8.28 (s, 1H), 7.99 (d, 1H), 7.85 (d, 2H), 7.61(d, 1H), 7.11 (d, 2H), 5.02 (s, 2H) and 3.80 (s, 3H) ppm.

4.2.ah6-(4-Methoxyphenylsulfinyl)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole6-(4-methoxyphenylsulfinyl)benzo[c][1,2]oxaborol-1(3H)-ol (C34)

¹H NMR (300 MHz, DMSO-d₆): δ 9.37 (s, 1H), 8.02 (d, 1H), 7.71 (dd, 1H),7.59 (d, 2H), 7.53 (d, 1H), 7.07 (d, 2H), 5.00 (s, 2H) and 3.76 (s, 3H)ppm.

4.2.ai 5-Trifluoromethyl-1,3-dihydro-1-hydroxy-2,1-benzoxaborole5-(trifluoromethyl)benzo[c][1,2]oxaborol-1(3H)-ol (C35)

M.p. 113-118° C. MS: m/z=203 (M+1) (ESI+) and m/z=201 (M−1) (ESI−).HPLC: 100% purity at 254 nm and 100% at 220 nm. ¹H NMR (300 MHz,DMSO-d₆): δ 9.48 (s, 1H), 7.92 (d, 1H), 7.78 (s, 1H), 7.67 (d, 1H) and5.06 (s, 2H) ppm.

4.2.aj 4-(4-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-4-yloxy)benzonitrile (C36)

For coupling reaction between 4-fluorobenzonitrile and substitutedphenol to give starting material 2, see Igarashi, S.; et al. Chemical &Pharmaceutical Bulletin (2000), 48(11), 1689-1697.

¹H-NMR (300 MHz, DMSO-d₆) (ppm) 4.84 (s, 2H), 7.08 (d, J=8.2 Hz, 2H),7.18 (d, J=7.9 Hz, 1H), 7.45 (t, J=7.3 Hz, 1H), 7.63 (d, J=7.3 Hz, 1H),7.82 (d, J=8.5 Hz, 2H).

4.2.ak 5-(3-Cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole3-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzonitrile (C37)

For coupling between 3-fluorobenzonitrile and substituted phenol to givestarting material 2: Li, F. et al., Organic Letters (2003), 5(12),2169-2171.

¹H-NMR (300 MHz, DMSO-d₆) (ppm) 4.93 (s, 2H), 7.0-7.1 (m, 2H), 7.3-7.4(m, 1H), 7.5-7.7 (m, 3H), 7.75 (d, J=8.2 Hz, 1H).

4.2.al 5-(4-Carboxyphenoxy)-1,3 dihydro-1-hydroxy-2,1-benzoxaborole4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)benzoic acid (C38)

To a solution of 5-(4-cyanophenoxy)-1-hydroxy-2,1-benzoxaborole obtainedin C17 (430 mg, 1.71 mmol) in ethanol (10 mL) was added 6 mol/L sodiumhydroxide (2 mL), and the mixture was refluxed for 3 hours. Hydrochloricacid (6 mol/L, 3 mL) was added, and the mixture was extracted with ethylacetate. The organic layer was washed with brine and dried on anhydroussodium sulfate. The solvent was removed under reduced pressure, and theresidue was purified by silica gel column chromatography (ethyl acetate)followed by trituration with diisopropyl ether to give the targetcompound (37 mg, 8%).

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.94 (s, 2H), 7.0-7.1 (m, 4H), 7.76(d, J=7.9 Hz, 1H), 7.94 (d, J=8.8 Hz, 2H), 9.19 (s, 1H), 12.8 (br s,1H).

4.2.am 1-Hydroxy-1,3dihydro-5-[4-(tetrazole-1-yl)phenoxy]-2,1-benzoxaborole5-(4-(1H-tetrazol-5-yl)phenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C39)

A mixture of 5-(4-cyanophenoxy)-1-hydroxy-2,1-benzoxaborole (200 mg,0.797 mmol), sodium azide (103 mg, 1.59 mmol), and ammonium chloride (85mg, 1.6 mmol) in N,N-dimethylformamide (5 mL) was stirred at 80° C. fortwo days. Water was added, and the mixture was extracted with ethylacetate. The organic layer was washed with water and brine, and dried onanhydrous sodium sulfate. The solvent was removed under reducedpressure, and the residue was purified by silica gel columnchromatography (ethyl acetate) followed by trituration with ethylacetate to give the target compound (55 mg, 23%).

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 4.95 (s, 2H), 7.0-7.1 (m, 2H), 7.23(d, J=8.8 Hz, 2H), 7.76 (d, J=7.9 Hz, 1H), 8.05 (d, J=8.5 Hz, 2H), 9.18(br s, 1H).

Example 5

Preparation of I from 2 Via 6

5.1 Catalytic Boronylation, Reduction and Cyclization

A mixture of 2 (10.0 mmol), bis(pinacolato)diboron (2.79 g, 11.0 mmol),PdCl₂ (dppf) (250 mg, 3 mol %), and potassium acetate (2.94 g, 30.0mmol) in 1,4-dioxane (40 mL) was stirred at 80° C. for overnight. Waterwas added, and the mixture was extracted with ethyl acetate. The organiclayer was washed with brine and dried on anhydrous sodium sulfate. Thesolvent was removed under reduced pressure. The crude product wasdissolved in tetrahydrofuran (80 mL), then sodium periodate (5.56 g,26.0 mmol) was added. After stirring at room temperature for 30 min, 2NHCl (10 mL) was added, and the mixture was stirred at room temperaturefor overnight. Water was added, and the mixture was extracted with ethylacetate. The organic layer was washed with brine and dried on anhydroussodium sulfate. The solvent was removed under reduced pressure, and theresidue was treated with ether to afford 6.3 mmol of the correspondingboronic acid. To the solution of the obtained boronic acid (0.595 mmol)in methanol (5 mL) was added sodium borohydride (11 mg, 0.30 mmol), andthe mixture was stirred at room temperature for 1 h. Water was added,and the mixture was extracted with ethyl acetate. The organic layer waswashed with brine and dried on anhydrous sodium sulfate. The solvent wasremoved under reduced pressure, and the residue was purified by silicagel column chromatography to give 0.217 mmol of I.

5.2 Results

Analytical data for exemplary compounds of structure I are providedbelow.

5.2.a 1,3-Dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole (C10)

Analytical data for this compound is listed in 4.2.j.

Example 6

Preparation of I from 3

6.1 One-pot Boronylation and Cyclization

To a solution of 3 (4.88 mmol) and triisopropyl borate (1.35 mL, 5.86mmol) in tetrahydrofuran (10 mL) was added n-butyllithium (1.6 mol/L inhexanes; 6.7 mL, 10.7 mmol) dropwise over 15 min at −78° C. undernitrogen atmosphere, and the mixture was stirred for 2 h while allowingto warm to room temperature. The reaction was quenched with 2N HCl, andextracted with ethyl acetate. The organic layer was washed with brineand dried on anhydrous sodium sulfate. The solvent was removed underreduced pressure, and the residue was purified by silica gel columnchromatography and treated with pentane to give 0.41 mmol of I.

6.2 Results

Analytical data for exemplary compounds of structure I are providedbelow.

6.2.a 1,3-Dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole (C10)

Analytical data for this compound is listed in 4.2.j.

Example 7

Preparation of I from 37.1 One-pot Boronylation and Cyclization with Distillation

To a solution of 3 (4.88 mmol) in toluene (20 mL) was added triisopropylborate (2.2 mL, 9.8 mmol), and the mixture was heated at reflux for 1 h.The solvent, the generated isopropyl alcohol and excess triisopropylborate were removed under reduced pressure. The residue was dissolved intetrahydrofuran (10 mL) and cooled to −78° C. n-Butyllithium (3.2 mL,5.1 mmol) was added dropwise over 10 min, and the mixture was stirredfor 1 h while allowing to warm to room temperature. The reaction wasquenched with 2N HCl, and extracted with ethyl acetate. The organiclayer was washed with brine and dried on anhydrous sodium sulfate. Thesolvent was removed under reduced pressure, and the residue was purifiedby silica gel column chromatography to give 1.54 mmol of I.

7.2 Results

Analytical data for exemplary compounds of structure I are providedbelow.

7.2.a 1,3-Dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole (C10)

Analytical data for this compound is listed in 4.2.j.

Example 8

Preparation of 8 from 7

8.1 Bromination

To a solution of 7 (49.5 mmol) in carbon tetrachloride (200 mL) wereadded N-bromosuccinimide (8.81 g, 49.5 mmol) and N,N-azoisobutylonitrile(414 mg, mol %), and the mixture was heated at reflux for 3 h. Water wasadded, and the mixture was extracted with chloroform. The organic layerwas washed with brine and dried on anhydrous sodium sulfate. The solventwas removed under reduced pressure to give the crude methyl-brominatedintermediate 8.

Example 9

Preparation of 3 from 8

9.1 Hydroxylation

To crude 8 (49.5 mmol) were added dimethylformamide (150 mL) and sodiumacetate (20.5 g, 250 mmol), and the mixture was stirred at 80° C. forovernight. Water was added, and the mixture was extracted with ether.The organic layer was washed with water and brine, and dried onanhydrous sodium sulfate. The solvent was removed under reducedpressure. To the residue was added methanol (150 mL) and 1N sodiumhydroxide (50 mL), and the mixture was stirred at room temperature for 1h. The reaction mixture was concentrated to about a third of volumeunder reduced pressure. Water and hydrochloric acid were added, and themixture was extracted with ethyl acetate. The organic layer was washedwith water and brine, and dried on anhydrous sodium sulfate. The solventwas removed under reduced pressure, and the residue was purified bysilica gel column chromatography followed by trituration withdichloromethane to give 21.8 mmol of 3.

9.2 Results

Exemplary compounds of structure 3 prepared by the method above areprovided below.

9.2.a 2-Bromo-5-cyanobenzyl Alcohol

¹H-NMR (300 MHz, DMSO-d₆) δ ppm 4.51 (d, J=5.9 Hz, 2H), 5.67 (t, J=5.6Hz, 1H), 7.67 (dd, J=8.2, 2.0 Hz, 1H), 7.80 (s, J=8.2 Hz, 1H), 7.83 (d,J=2.0 Hz, 1H).

Additional examples of compounds which can be produced by this methodinclude 2-bromo-5-(4-cyanophenoxy)benzyl alcohol.

Example 10

Preparation of 9 from 2

10.1 Reaction

A mixture of 2 (20.0 mmol), (methoxymethyl)triphenylphosphonium chloride(8.49 g, 24.0 mmol), and potassium tert-butoxide (2.83 g, 24.0 mol) inN,N-dimethylformamide (50 mL) was stirred at room temperature forovernight. The reaction was quenched with 6 N HCl, and the mixture wasextracted with ethyl acetate. The organic layer was washed with water(×2) and brine, and dried on anhydrous sodium sulfate. The solvent wasremoved under reduced. To the residue were added tetrahydrofuran (60 mL)and 6 N HCl, and the mixture was heated at reflux for 8 h. Water wasadded, and the mixture was extracted with ether. The organic layer waswashed with brine and dried on anhydrous sodium sulfate. The solvent wasremoved under reduced pressure to afford 16.6 mmol of 9.

Example 11 Preparation Method of Step 13 11.1 Reaction

A solution of I in an appropriate alcohol solvent (R¹—OH) was refluxedunder nitrogen atmosphere and then distilled to remove the alcohol togive the corresponding ester.

Example 12

Preparation of Ib from Ia

12.1 Reaction

To a solution of Ia in toluene was added amino alcohol and theparticipated solid was collected to give Ib.

12.2 Results

(500 mg, 3.3 mmol) was dissolved in toluene (37 mL) at 80° C. andethanolamine (0.20 mL, 3.3 mmol) was added. The mixture was cooled toroom temperature, then ice bath, and filtered to give C40 as a whitepowder (600.5 mg, 94%).

12.2a Ethanolamine adduct of1,3-Dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole (C40)

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 2.88 (t, J=6.2 Hz, 2H), 3.75 (t, J=6.3Hz, 2H), 4.66 (s, 2H), 5.77 (br, 2H), 6.85-6.91 (m, 2H), 7.31 (td,J=7.2, 1.2 Hz, 1H).

Example 13 Formulations

Compounds of the present invention can be administered to a patientusing a therapeutically effective amount of a compound described hereinin any one of the following three lacquer formulations and one solventformulation. The lacquer formulation provides good durability while thesolvent formulation provides good ease of use. These compounds can alsobe applied using a spray formulation, paint-on lacquer, drops, or other.

-   -   1. 1:4 propylene glycol:ethanol; 1:10 wt/vol compound of        invention;    -   2. 1:4 poly(vinyl methyl ether-alt-maleic acid monobutyl ester:        ethanol; 1:10 wt/vol compound of the invention;    -   3. 56% ethanol; 14% water; 15% poly(2-hydroxyethyl        methacrylate); 5% dibutyl sebacate; 10% compound of the        invention;    -   4. 55% ethanol; 15% ethyl acetate; 15% poly(vinyl acetate); 5%        dibutyl sebacate; 10% compound of the invention.

The preparation of these formulations is well known in the art and isfound in references such as Remington: The Science and Practice ofPharmacy, supra.

Example 14 Antifungal MIC Testing

All MIC testing followed the National Committee for Clinical LaboratoryStandards (NCCLS) guidelines for antimicrobial testing of yeasts (M27-A2NCCLS) and filamentous fungi (Pfaller et al., NCCLS publicationM38-A—Reference Method for Broth Dilution Antifungal SusceptibilityTesting of Filamentous Fungi; Approved Standard. Wayne, Pa.: NCCLS; 2002(Vol. 22, No. 16) except the Malassezia species which was incubated in aurea broth (Nakamura et al., Antimicrobial Agents And Chemotherapy,2000, 44(8) p. 2185-2186). Results of the MIC testing is provided inFIG. 1.

Example 15 Keratin Assay

Many antifungal agents strongly bind to keratin which not only reducestheir antifungal potency but also may restrict their penetration intothe nail. The affinities of the compounds for keratin powder wasdetermined by a method described in Tatsumi, Antimicrobial Agents andChemotherapy, 46(12):3797-3801 (2002).

A comparison of MIC data for several compounds of the invention againstT. rubrum, with and without the presence of 5% keratin, is provided inFIG. 1.

Example 16

(C10) Antifungal Spectrum of Activity

(C10) is a novel compound in development for use as a topical antifungaltreatment. The purpose of this study was to determine the minimuminhibitory concentration (MIC) for (C10) against 19 test strains offungi including: Aspergilus fumigatus (A. fumigatus), Candida Albicans(C. albicans, both fluconazole sensitive and resistant strains), Candidaglabrata (C. glabrata), Candida krusei (C. krusei), Cryptococcusneoformans (C. neoformans), Candida parapsilosis (C. parapsilosis),Candida tropicalis (C. tropicalis), Epidermophyton floccosum (E.floccosum), Fusarium solani (F. solani), Malassezia furfur (M. furfur),Malassezia pachydermatis (M. pachydermatis), Malassezia sympodialis (M.sympodialis), Microsporum audouinii (M. audouinii), Microsporum canis(M. canis), Microsporum gypseum (M. gypseum), Trichophytonmentagrophytes (T. mentagrophytes), Trichophyton rubrum (T. rubrum),Trichophyton tonsurans (T. tonsurans). Fungal growth was evaluated afterexposure to different concentrations of (C10). In addition, the MIC for(C10) against T. rubrum in the presence of 5% keratin powder and theminimum fungicidal concentration (MFC) for (C10) against T. rubrum andT. mentagrophytes were also determined. Ciclopirox and/or terbinafineand/or fluconazole and/or itraconazole were used as comparators andtested in a similar manner. These studies were conducted at NAEJAPharmaceutical, Inc.

Materials and Methods

(C10) was obtained from Anacor Pharmaceuticals, Inc. (Palo Alto, Calif.,USA). ATCC strains were obtained from ATCC (Manassas, Va., USA).Ciclopirox-olamine was obtained from Sigma-Aldrich Co. (St. Louis, Mo.,USA). Terbinafine, fluconazole and itraconazole were synthesized atNAEJA Pharmaceutical Inc. (Edmonton, AB, Canada), experimentalprocedures and analytical data for these standards are stored in NAEJAarchives.

All MIC testing followed the National Committee for Clinical LaboratoryStandards (NCCLS) guidelines for antimicrobial testing of yeasts andfilamentous fungi (Pfaller et al., 2002) except the Malassezia specieswhich were incubated in a urea broth (Nakamura et al., 2000). Themicrobroth dilution method was used to test the in vitro activity of(C10) against 19 test strains of fungi. Briefly, compounds weredissolved in DMSO and diluted in sterile water to give a working stock.Two-fold serial dilutions of the working stock were prepared in 96-wellplates and media was added. Media was RPMI, RPMI+MOPS, modified RPMI, ormodified Urea broth. The plates were inoculated with the fungalsuspensions to give a final inoculum size of 0.5−2.5×10³ cells/mL foryeasts or 0.4−5×10⁴CFU/mL for filamentous fungi and then incubated for24-168 h at 35° C. The final concentration of DMSO did not exceed 5%.The MIC was defined as the lowest concentration that resulted in over90% reduction of growth, as compared to a drug-free control. The MFC wasdefined as the lowest concentration that killed over 90% of the fungi,as compared to a drug-free control.

Results and Conclusions

The results for the MIC of (C10) and reference compounds against 19strains of fungi are shown in FIG. 2. The results for the MFC of C10against 2 strains of fungi are shown in Table 2. (C10) had MIC valuesranging from 0.25-2 g/mL against all fungi tested. Addition of 5%keratin powder to the media did not effect the MIC against T. rubrum.(C10) had fungicidal activity against T. rubrum and T. mentagrophyteswith MFC values of 8 and 16 g/mL, respectively. Reference compounds hadMIC values in the range defined by NCCLS.

Example 17 The Solubility, Stability and Log P Determination ofCompounds of the Present Invention by LC/MS/MS

The solubility, room temperature stability and Log P of C10 wasdetermined by the following methodology.

Reagents and Standards:

Ethanol: 200 proof ACS Grade (EM Science, Gibbstown, N.J., USA);Octanol: Octyl alcohol (EM Science, Gibbstown, N.J., USA); Acetonitrile:HPLC Grade (Burdick & Jackson, Muskegon, Mich., USA); Ammonium Acetate:lot 3272×49621 (Mallinckrodt, Phillipsburg, N.J., USA); C10: lotA032-103 (Anacor Pharmaceuticals, Palo Alto, Calif., USA); p-Nitrophenol(PNP): lot OGNO1 (TCI America, Portland, Oreg., USA); Water: Deionizedwater (from Millipore systems, Billerica, Mass., USA)

Solubility

N-Octanol and water were mutually pre-saturated by vigorously stirring amixture of both solvents for up to 12 h and the mixture was allowed toseparate. Solubility in each solvent was determined by adding 10 μL of20, 40, 200, 1000 and 5000 g/mL of C10 in DMSO to the pre-saturatedn-octanol or water. After the sample was vortexed for 10 sec, the samplewas centrifuged for 10 min at ca. 3000 rpm. A visual inspection was madeto determine if the sample was clear or if a pellet had formed on thebottom of the tube.

Log P

C10 (10 μL of 5000 μ/mL) at 2× the final concentration was added to 0.5mL pre-saturated n-octanol and mixed. An equal volume (0.5 mL) ofpre-saturated water was added, vortex mixed and then mixed on a rotatingshaker for one hour and 24 h in triplicate at ca. 25° C. The organic andaqueous layers were separated by centrifugation for 5 min at ca. 2000rpm. Twenty five L of the octanol (top) layer were removed and placed ina pre-labeled tube. Twenty five L of the aqueous layer (bottom) wereremoved, taking care to avoid octanol contamination, and placed in apro-labeled tube.

Stability at Room Temperature

C10 (10 μL of 5000 g/mL) was added both to 0.5 mL n-octanol and 0.5 mLwater in triplicate. Samples were mixed. At 0 h and 24 h samples werestored at ca. −20° C. Twenty five μL of sample was used for analysis.

Extraction Procedure C10

For the octanol sample, 25 μL of ethanol, 25 μL of water and 300 μL ofacetonitrile containing the internal standard was added. For the watersample, 25 μL of ethanol, 25 μL of octanol and 300 μL of acetonitrilecontaining the internal standard [60 mL of acetonitrile add 6 μL of PNP(1000 μg/mL)] was added. For the calibrators 25 μL of octanol, 25 μL ofwater and 300 pL of acetonitrile containing the internal standard wasadded. The sample was vortexed for 10 seconds. Two hundred μL of theorganic layer were transferred into a clean deactivated autosamplervial.

Calculations

A l/concentration weighted linear regression was used for thequantitation of C10. All integration were performed with peak areasusing Analyst version 1.3, Applied Biosystems. For C10, peak area ratiosanalyte to internal standard PNP were used for all quantitation.

The partition coefficient (P) was calculated according to the equationdetailed below:

P=[Sample concentration]_(octanol)/[Sample concentration]_(water)

Log P=log₁₀(partition coefficient)

Results:

As shown in Table 17A the solubility of C10 in both octanol and water isvery good over the concentration range tested.

TABLE 17A Solubility of C10 in water and octanol Targeted Conc WaterOctanol (μg/mL) Visual Visual 0.800 Clear Clear 4.00 Clear Clear 20.0Clear Clear 100 Clear Clear

Table 17B shows the results of the log P determination after 1 h and 24h for C10. The mean log P after 1 h was 1.97 (n=3). After 24 h theconcentrations in both the octanol and water layer remained the same.The mean log P after 24 h was 1.93 (n=3).

TABLE 17B Log P of C10 Conc. in Water Conc. in Octanol Sample (μg/mL)(μg/mL) Log P  1 h-1 1.26 108 1.93  1 h-2 1.21 103 1.93  1 h-3 1.05 1152.04 24 h-1 1.27 104 1.91 24 h-2 1.17 109 1.97 24 h-3 1.28 99.0 1.89

A stability study for C10 was initiated at room temperature over 24 hwithout continuous mixing. Table 17C shows that C10 in pure water andoctanol is stable over 24 h.

TABLE 17C Water and Octanol stability for C10 at room temperature after24 h. Percent Mean Remaining 24 h Sample (μg/mL) SD versus 0 g Water-0 h82.5 3.72 115 Water-24 h 95.0 21.4 Octanol-0 h 115 3.06 93 Octanol-24 h107 6.11

Example 18

Determination of Penetration of C10 into the Human Nail

Two nail penetration studies were performed based on the protocol in Huiet al., Journal of Pharmaceutical Sciences, 91(1): 189-195 (2002) (“Huiprotocol”). The purpose of this study was to determine and compare thepenetration and distribution of C10 in vehicle into the human nail platein vitro relative to 8% ciclopirox w/w in commercial lacquer (Penlac®).

Materials and Methods

Test Article and Dosage Formulation

8% ciclopirox w/w in commercial lacquer was manufactured by Dermick(Berwyn, Pa.). The radiochemical purity and specific activity of thechemical was determined as >95% and 12.5 mCi/mmol, respectively.

The study was composed of two groups. The compositions (weight %) of thedosage formulations are as follows:

Active radiolabeled compound in four groups.

Dosing Test Chemical Radioactivity Groups* (×14 days) (%) (per 10 μL) A(C10) qd 10 0.19 μCi C (Ciclopirox) qd 8 0.22 μCi *A = C10 group, C =Ciclopiriox group

Human Nails

Healthy human finger nail plates were collected from adult humancadavers and stored in a closed container at 0-4° C. Before theexperiment, the nail plates were gently washed with normal saline toremove any contamination, then re-hydrated by placing them for threehours on a cloth wetted with normal saline. The nail samples wererandomly selected into four groups.

Dosing and Surface Washing Procedures

Dose Preparation:

Radioactivity of each group is approximately 0.19±0.01 and 0.22±0.03μCi/10 μL solutions respectively, for ¹⁴C—C10 (group A), and¹⁴C-ciclopirox (group C).

Experiment Procedure:

Study Group A Group C Day wash dose sample wash dose sample 1 D D 2 W DW D 3 W D C W D C 4 W D W D 5 W D W D 6 W D C W D C 7 W D W D 8 W D W D9 W D C W D C 10 W D W D 11 W D W D 12 W D C W D C 13 W D W D 14 W D W D15 W C, N W C, N W = once per day before dosing (9~10 AM). D = once perday (9~10 AM). C = changing/sampling cotton ball after surface washingbefore topical dosing. N = Nail sampling.

Washing Procedure

Surface washing was started in morning 10 min prior to next dosing, thesurface of the nail was washed with cotton tips in a cycle, as follows:

a tip wetted with absolute ethanol, then

a tip wetted with absolute ethanol, then

a tip wetted with 50% IVORY liquid soap, then

a tip wetted with distilled water, then

a final tip wetted with distilled water.

The washing samples from each cycle of each nail were pooled andcollected by breaking off the cotton tip into scintillation glass vials.Aliquots of 3.0 mL methanol were added into each vial to extract testmaterial. The radioactivity of each sample was measured in a liquidscintillation counter.

Incubation System

A Teflon one-chamber diffusion cell (PermeGear, Inc., Hellertown, Pa.)was used to hold each nail. To approximate physiological conditions, asmall cotton ball wetted with 0.1 mL normal saline was placed in thechamber to serve as a nail bed and provide moisture for the nail plate.Every 3 days, 0.1 mL normal saline was injected through the inlet intothe chamber to keep the cotton ball wet. The nail plate was placed on aledge inside the receptor (1.0 cm in diameter and 0.5 cm high). Theventral (inner) surface of the nail was placed face down and rested onthe wet cotton ball. The cells were placed on a platform in a largeglass holding tank filled with saturated sodium phosphate solution tokeep the cells at a constant humidity of 40%.

Sampling Instrument

The nail sampling instrument had two parts, a nail sample stage and adrill. The nail sampling stage consists of a copper nail holder, threeadjustments, and a nail powder capture. Three adjustments allow movementin vertical direction. The first coarse adjustment (on the top) was forchanging the copper cell and taking powder samples from the capture. Theother two adjustments (lower) were for sampling process. The secondcoarse adjustment allowed movement of 25 mm and the fine adjustmentprovides movement of 0.20 mm. The nail powder capture was locatedbetween the copper cell and the cutter. The inner shape of the capturewas inverted funnel and the end of funnel connects to a vacuum. Byplacing a circle filter paper inside of the funnel, the nail powdersamples were captured on the filter paper during the sampling process.

Sampling Procedure

After completion of the incubation phase, the nail plate was transferredfrom the diffusion cell to a clean copper nail holder for samplingprocess. The nail plate was inverted so that the ventral (nail bed)surface now faced up and the dorsal (outer) dosed surfaced faced down.The copper nail holder has an opening as it sits on top of the stage.When the sampling process initiated, the coarse adjustment was adjustedto move the position of the stage until the nail plate was just touchingthe tip of the cutter. Then the drill was turned on and the fineadjustment was turned to push the stage closer to the drill, removing anail core sample. After the above process, approximate 0.40-0.50 mm indepth and 7.9 mm in diameter nail pulverized samples were harvested fromthe center of the ventral (nail bed) surface of the nail.

The powdered nail samples were collected into a glass scintillation vialand weighted. Aliquots of 5.0 mL Packard soluene-350 (Packard InstrumentCompany, Meriden, Conn.) was added to the scintillation vial to dissolvethe powder. The upper part, the intermediate and dorsal layers of thecenter of the nail, including the area of application of the dose wascut in the same diameter as the sampled area and was then placed into aglass scintillation vial with 5.0 mL packard soluene-350. The rest ofthe nail was also placed in a glass scintillation vial with 5.0 mLpackard soluene-350.

The amount of nail sample removed was measured by the difference inweight of the nail plate before and after drilling, and collecting thecore of powder.

Radioactivity Measurement

All radioactivity measurements were conducted with a Model 1500 LiquidScintillation Counter (Packard Instrument Company, Downer Grove, Ill.).The counter was audited for accuracy using sealed samples of quenchedand unquenched standards as detailed by the instrument manual. The ¹⁴Ccounting efficiency is equal to or greater than 95%. All nail samplespre-treated with packard soluene-350 were incubated at 40° C. for 48hours followed by the addition of 10 mL scintillation cocktail(HIONIC-FLUOR, Packard Instrument Company, Meriden, Conn.). Othersamples (standard dose, surface washing, and bedding material) weremixed directly with Universal ES scintillation cocktail (ICNBiomedicals, Costa Mesa, Calif.). Background control and test sampleswere counted for 3 minutes each for radioactivity.

Data Analysis

All sample counts (expressed as dpm) were transcribed by hand to acomputerized spreadsheet (Microsoft Excel). The individual and mean(±S.D.) amount of test chemical equivalent in nail, bedding material,and wash samples are presented as dpm, μCi, percent administered dose,and mg equivalent at each time point. The concentration of ¹⁴C-labeledtest chemicals were calculated from the value based on the specificactivity of each [¹⁴C]-test chemical. The information of concentrationof non-labeled test chemical in the topical formulation was obtainedfrom the manufactures. Total concentration of test chemical equivalentis the sum of the concentration of ¹⁴C-labeled test chemical and theconcentration of non-labeled test chemical. The value of total amount oftest chemical equivalent in each nail sample was calculated from thosevalues based on radioactivity of the sample and the ratio of total mgtest chemical equivalent and radioactivity of the test chemical. Thedata was further normalized by dividing with the weight of the sample.Statistical significant of nail samples from every two groups wasanalyzed by student t-test.

Results Characteristics of Nail Samples

For both groups (Group A group and Group C) the thickness of whole nailplate, the depth of the ventral surface core sample removed by cutter,the percentage of the whole nail thickness, and the actual weight ofpowdered nail sample were collected. No statistical difference is foundbetween two groups (P>0.05).

Weight Normalized C10 and Ciclopirox Equivalent in Nail

FIG. 3 shows summarized normalized drug equivalents in each part (layer)of nail samples. After weight normalization, the concentration of C10equivalent in dorsal/intermediate center, ventral/intermediate center,and remainder nail samples was significantly higher than that ofciclopirox equivalent (p<0.002).

C10 and Ciclopirox Equivalent in Cotton Ball Nail Supporting Bed

FIG. 4 shows summarized C10 and ciclopirox equivalent in supporting bedcotton ball samples. Similar to weight normalized C10 equivalent in thenail plate samples, absolute amount of C10 equivalent per cotton ballsample in group A (after 14 day dosing) was significantly higher thanthat of ciclopirox in group C (p≦0.004). The difference of these twotest chemicals was 250 times.

Mass Balance of Radioactivity of [¹⁴C]-C10 and [¹⁴C]-Ciclopirox after14-Day Treatment

Table 5 shows summarized radioactive recovery from washing, nailsamples, and supporting bed cotton ball samples. Cumulativeradioactivity recoveries of carbon-14 were 88±9.21, and 89±1.56 percentof applied dose in group A, and group C, respectively. 88% of theradiolabeled material was accounted for.

Conclusion

In this study, penetration rate of [¹⁴C]-C10 in Anacor topicalformulation and [¹⁴C]-ciclopirox (8% w/w in commercial lacquer) intohuman nail with four different dosing and washing methods was studied.

Results show that much more amount of [¹⁴C]-C10 penetrating into thedeeper parts of the nail when compared with [¹⁴C]-ciclopirox. Tables 3and 4 show that the amount of [¹⁴C]-C10 equivalent inventral/intermediate center of the nail layer and cotton ball supportingbed in the group A was statistically higher (p≦0.002) than group C aftera 14-day dosing period.

Example 19

Determination of Penetration of CO into the Human Nail

The aim of the current study was to assess and compare the perungualabsorption of C10 in a simple vehicle using MedPharm's TurChub® model(see http://www.medpharm.co.uk; specificallyhttp://www.medpharm.co.uk/downloads/Skin%20and%20nail%20dec%202003.pdf;viewed Feb. 14, 2006). in a full scale experiment. Six replicatesinvolving C10 were conducted and Formulations Y (8% ciclopirox w/w incommercial lacquer) and Z (Loceryl, 5% amorolfine w/v in commerciallacquer) were used as the reference formulations.

The following materials were used in these experiments. These materialswere used without any modifications.

A dose of 40 μL/cm² of the test compound C10 in 50:50 propyleneglycol:ethyl acetate was applied to a full thickness nail sample eachday over a total duration of five days. Both the reference formulationswere also applied at the same dose.

TurChub® Zone of Inhibition Experiment

Placebo, test item C10 in vehicle and the reference formulations Y and Zwere tested for their inhibition of Trichophyton rubrum (T. rubrum)growth after penetration through a full thickness human nail using azone of inhibition measurement.

Formulation Efficacy Testing

FIGS. 5-9 show the results obtained from the TurChub zone of inhibitionassays. It can be observed that C10 is a potent antifungal agent, whichcan penetrate through a full thickness nail to elicit its effect againstthe target organism T. rubrum. No zones of inhibition were observed withreference formulations Y and Z or with the placebo for C10. Theexperiment using C10 was repeated for a second time to confirm theresult and it can be observed from FIGS. 6 and 7 that C10 shows zones ofinhibition of 100%, 67%, 46%, 57%, 38% and 71% in the first experimentand 74%, 86%, 100%, 82%, 100% and 84% in the second experiment. Themeasurement was taken from the nail to the first point of growthobserved.

From the results obtained using MedPharm's TurChub zone of inhibitionassay as a test system, the test item C10 was found to be a powerfulantifungal agent and demonstrated superior results vs. the commercialreference formulations Y and Z. From these experiments it appears thatthe compound is permeating through a full thickness nail barrier toexhibit the antifungal activity.

Example 20

Determination of Penetration of C10 into the Human Nail: Dose Response

The optimal dose-response range for penetration into the human nail wasdetermined to be between 1% and 15%. The experiments to determine theoptimal dose-response was conducted as follows.

Tests at different test compound concentrations were conducted on nailsderived from the same cadaver. Cadaver nails were hydrated overnight,cut into 4 equally sized squares and placed onto individual poloxomersupports. Test articles were formulated in a lacquer at 1%, 2.5%, 5%,7.5%, 10% and 15% w/v. A 40 μL/cm² dose is applied to the center of thenail piece and the nails are left for 24 hrs. Nails are removed from thepoloxomer support. Poloxomer support is analyzed for quantity ofcompound using LC/MS/MS.

Example 21 Preparation of Pyridinyloxaboroles

21a. Metallation and Boronylation

To a solution of 3-bromo-4-hydroxymethylpyridine (10.7 mmol) and B(OMe)₃(2.73 mL, 11.9 mmol) in anhydrous THF (20 mL) at −78° C. under nitrogenwas added dropwise n-BuLi (13.6 mL, 21.8 mmol). The cooling bath wasthen removed. The mixture was warmed gradually with stirring for 30 minand then stirred with a water bath for 2 h. Brine was then added and thepH adjusted to 7 using 6N HCl. The mixture was washed with THF (×2) andthe aqueous layer (containing product) was evaporated to dryness. Theresidue was washed with THF and the product was extracted into ethanol(×2). Ethanol was removed in vacuo, water was added to the residue andremoved in vacuo. Toluene was added and removed in vacuo. The resultingresidue was triturated with diethyl ether and the product was collectedby filtration to afford C12.

21b.7-Hydroxy-2,1-oxaborolano[5,4-c]pyridine[[1,2]oxaborolo[3,4-c]pyridin-1(3H)-ol](C12)

¹H-NMR (300 MHz, DMSO-d₆): δ ppm 5.00 (s, 2H), 7.45 (d, J=5.0 Hz, 1H),8.57 (d, J=5.3 Hz, 1H), 8.91 (s, 1H), 9.57 (s, 1H). ESI-MS m/z 134(M-H)⁻, C₆H₆BNO₂=135.

Example 22 Cyclic Borinic Esters

Additional compounds can be produced by the methods described herein. Bychoosing the appropriate starting material such as 1 or 3, Examples 1-7can be used to formulate the following compounds. Where available,melting point characterization is provided for these compounds.

22. Results

Analytical data for exemplary compounds of structure I are providedbelow.

22a Ethyl 2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)acetate(C41)

M.P. 134-137° C. Exemplary starting material: ethyl2-(4-bromo-3-(hydroxymethyl)phenoxy)acetate.

22b 2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)acetic acid(C42)

M.P. 163-166° C. Exemplary starting material: ethyl2-(4-bromo-3-(hydroxymethyl)phenoxy)acetate. The title compound isobtained after saponification of the corresponding ester.

22c 6-(thiophen-2-ylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C43)

M.P. 99-104° C. Exemplary starting material:(2-bromo-4-(thiophen-2-ylthio)phenyl)methanol.

22d 6-(4-fluorophenylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C44)

M.P. 135-138° C. Exemplary starting material:(2-bromo-4-(4-fluorophenylthio)phenyl)methanol.

22e1-(3-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)methyl)phenyl)pentan-1-one(C45)

M.P. 96-98° C. Exemplary starting material:1-(3-((4-bromo-3-(hydroxymethyl)phenoxy)methyl)phenyl)pentan-1-one.

22f2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)-1-(piperidin-1-yl)ethanone(C46)

M.P. 158-163° C. Exemplary starting material:2-(4-bromo-3-(hydroxymethyl)phenoxy)-1-(piperidin-1-yl)ethanone.

22g2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)ethanone(C47)

M.P. 190-195° C. Exemplary starting material:2-(4-bromo-3-(hydroxymethyl)phenoxy)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)ethanone.

22h 6-(4-(pyridin-2-yl)piperazin-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol(C48)

M.P. 135-138° C. Exemplary starting material:(2-bromo-4-(4-(pyridin-2-yl)piperazin-1-yl)phenyl)methanol.

22i 6-nitrobenzo[c][1,2]oxaborol-1(3H)-ol (C49)

M.P. 163-171° C. Exemplary starting material:benzo[c][1,2]oxaborol-1(3H)-ol. See JACS 82, 2172, 1960 for preparation.

22j 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol (C50)

M.P. 145-148° C. Exemplary starting material:6-nitrobenzo[c][1,2]oxaborol-1(3H)-ol.

22k 6-(dimethylamino)benzo[c][1,2]oxaborol-1(3H)-ol (C51)

M.P. 120-123° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22l N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)benzamide (C52)

M.P. 186-193° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22m 6-(4-phenylpiperazin-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (C53)

M.P. 159-161° C. Exemplary starting material:(2-bromo-4-(4-phenylpiperazin-1-yl)phenyl)methanol.

22o 6-(1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (C55)

M.P. 135-140° C. Exemplary starting material:(2-bromo-4-(1H-indol-1-yl)phenyl)methanol.

22p 6-morpholinobenzo[c][1,2]oxaborol-1(3H)-ol (C56)

M.P. 128-132° C. Exemplary starting material:(2-bromo-4-morpholinophenyl)methanol.

22q6-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)nicotinonitrile(C57)

M.P. 193-198° C. Exemplary starting material:6-(4-bromo-3-(hydroxymethyl)phenoxy)nicotinonitrile.

22r 5-fluoro-6-nitrobenzo[c][1,2]oxaborol-1(3H)-ol (C58)

M.P. 162-167° C. Exemplary starting material:5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol.

22s 5-bromo-6-(hydroxymethyl)benzo[c][1,2]oxaborol-1(3H)-ol (C59)

M.P.>257° C. Exemplary starting material:(2,5-dibromo-4-(methoxymethyl)phenyl)methanol.

22t 3,7-dihydro-1,5-dihydroxy-1H,3H-Benzo[1,2-c:4,5-c′]bis[1,2]oxaborole(C60)

M.P.>250° C. Exemplary starting material:(2,5-dibromo-1,4-phenylene)dimethanol.

22u 1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-3-phenylurea(C61)

M.P. 213-215° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22vN-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)benzenesulfonamide(C62)

M.P. 175-184° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22w N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)acetamide (C63)

M.P. 176-185° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22x 7-(hydroxymethyl)benzo[c][1,2]oxaborol-1(3H)-ol (C64)

M.P. 241-250° C. Exemplary starting material:(2-bromo-1,3-phenylene)dimethanol.

22y 7-methylbenzo[c][1,2]oxaborol-1(3H)-ol (C65)

M.P. 107-111° C. Exemplary starting material:(2-bromo-3-methylphenyl)methanol.

22z 6-(3-(phenylthio)-1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (C66)

M.P. 159-163° C. Exemplary starting material:(2-bromo-4-(3-(phenylthio)-1H-indol-1-yl)phenyl)methanol.

22aa3-(1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-1H-indol-3-ylthio)propanenitrile(C67)

M.P. 135-141° C. Exemplary starting material:3-(1-(3-bromo-4-(hydroxymethyl)phenyl)-1H-indol-3-ylthio)propanenitrile.

22bb 6-(5-methoxy-1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (C68)

M.P. 120-124° C. Exemplary starting material:(2-bromo-4-(5-methoxy-1H-indol-1-yl)phenyl)methanol.

22cc 5,6-methylenedioxybenzo[c][1,2]oxaborol-1(3H)-ol. (C69)

M.P. 185-189° C. Exemplary starting material:(6-bromobenzo[d][1,3]dioxol-5-yl)methanol.

22dd 6-amino-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C70)

M.P. 142-145° C. Exemplary starting material:6-nitro-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol.

22ee 6-(benzylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C71)

M.P. 159-164° C. Exemplary starting material:6-amino-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol.

22ff6-(5-methoxy-3-(phenylthio)-1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol(C72)

M.P. 135-141° C. Exemplary starting material:(2-bromo-4-(5-methoxy-3-(phenylthio)-1H-indol-1-yl)phenyl)methanol.

22gg3-(1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-5-methoxy-1H-indol-3-ylthio)propanenitrile(C73)

M.P. 149-154° C. Exemplary starting material:3-(1-(3-bromo-4-(hydroxymethyl)phenyl)-5-methoxy-1H-indol-3-ylthio)propanenitrile.

22hh 4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-7-yloxy)benzonitrile(C74)

M.P. 148-153° C. Exemplary starting material:4-(2-bromo-3-(hydroxymethyl)phenoxy)benzonitrile.

22ii 6-(5-chloro-1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (C75)

M.P. 149-154° C. Exemplary starting material:(2-bromo-4-(5-chloro-1H-indol-1-yl)phenyl)methanol.

22jj3-(5-chloro-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-1H-indol-3-ylthio)propanenitrile(C76)

M.P.>225° C. Exemplary starting material:3-(1-(3-bromo-4-(hydroxymethyl)phenyl)-5-chloro-1H-indol-3-ylthio)propanenitrile.

22kk 6-(benzylamino)benzo[c][1,2]oxaborol-1(3H)-ol (C77)

M.P. 126-133° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22ll 6-(dibenzylamino)benzo[c][1,2]oxaborol-1(3H)-ol (C78)

M.P. 115-123° C. Exemplary starting material:6-aminobenzo[c][1,2]oxaborol-1(3H)-ol.

22mm 7-(4-(1H-tetrazol-5-yl)phenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C79)

M.P. decomposition at 215° C. Exemplary starting material:4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-7-yloxy)benzonitrile.

22nn6-(5-chloro-3-(phenylthio)-1H-indol-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol(C80)

M.P. 145-151° C. Exemplary starting material:(2-bromo-4-(5-chloro-3-(phenylthio)-1H-indol-1-yl)phenyl)methanol.

22pp 6-(4-(pyrimidin-2-yl)piperazin-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol(C82)

M.P. NA ° C. Exemplary starting material:(2-bromo-4-(4-(pyrimidin-2-yl)piperazin-1-yl)phenyl)methanol.

22qq 7-(benzyloxy)benzo[c][1,2]oxaborol-1(3H)-ol (C83)

M.P. NA ° C. Exemplary starting material:(3-(benzyloxy)-2-bromophenyl)methanol.

22rr 4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-ylthio)pyridiniumchloride (C84)

M.P. NA ° C. Exemplary starting material:(2-bromo-4-(pyridin-4-ylthio)phenyl)methanol.

22ss 6-(pyridin-2-ylthio)benzo[c][1,2]oxaborol-(3H)-ol (C85)

M.P. NA ° C. Exemplary starting material:(2-bromo-4-(pyridin-2-ylthio)phenyl)methanol.

22tt 7-fluorobenzo[c][1,2]oxaborol-1(3H)-ol (C86)

M.P. 120-124° C. Exemplary starting material:(2-bromo-3-fluorophenyl)methanol.

22uu 6-(4-(trifluoromethyl)phenoxy)benzo[c][1,2]oxaborol-1(3H)-ol (C87)

M.P. 98-105° C. Exemplary starting material:(2-bromo-4-(4-(trifluoromethyl)phenoxy)phenyl)methanol.

22vv 6-(4-chlorophenylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C88)

M.P. 157-161° C. Exemplary starting material:(2-bromo-4-(4-chlorophenylthio)phenyl)methanol.

22ww 6-(4-chlorophenylsulfinyl)benzo[c][1,2]oxaborol-1(3H)-ol (C89)

M.P. 154-161° C. Exemplary starting material:6-(4-chlorophenylthio)benzo[c][1,2]oxaborol-1(3H)-ol.

22xx 6-(4-chlorophenylsulfonyl)benzo[c][1,2]oxaborol-1(3H)-ol (C90)

M.P. 157-163° C. Exemplary starting material:6-(4-chlorophenylthio)benzo[c][1,2]oxaborol-1(3H)-ol.

22yyN-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)-N-(phenylsulfonyl)benzenesulfonamide(C91)

M.P. 142-152° C. Exemplary starting material:N-(4-bromo-3-(hydroxymethyl)phenyl)-N-(phenylsulfonyl)benzenesulfonamide.

22zz 6-(4-(trifluoromethyl)phenylthio)benzo[c][1,2]oxaborol-1(3H)-ol(C92)

M.P. 111-113° C. Exemplary starting material:(2-bromo-4-(4-(trifluoromethyl)phenylthio)phenyl)methanol.

22aaa6-(4-(trifluoromethyl)phenylsulfinyl)benzo[c][1,2]oxaborol-1(3H)-ol(C93)

M.P. 79-88° C. Exemplary starting material:6-(4-(trifluoromethyl)phenylthio)benzo[c][1,2]oxaborol-1(3H)-ol.

22bbb 6-(4-(methylthio)phenylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C94)

M.P. 117-120° C. Exemplary starting material:(2-bromo-4-(4-(methylthio)phenylthio)phenyl)methanol.

22 ccc 6-(p-tolylthio)benzo[c][1,2]oxaborol-1(3H)-ol (C95)

M.P. 139-144° C. Exemplary starting material:(2-bromo-4-(p-tolylthio)phenyl)methanol.

22ddd3-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yloxy)methyl)benzonitrile(C96)

M.P. 147-150° C. Exemplary starting material:3-((4-bromo-3-(hydroxymethyl)phenoxy)methyl)benzonitrile.

Example 23

Alternative Preparation of 4 from 3

A 22.0 L 3-neck flask was equipped with a stir motor, N₂ inlet, additionfunnel, heating mantle, and condenser. The flask was charged with 3500 g(17.1 mol) of 2-bromo-5-fluorobenzyl alcohol followed by the addition of3556 g of tetrahydrofuran and 16.4 g (0.17 mol) of methanesulfonic acid.Next, 400 g (4.7 mol) of 3,4-dihydro-2H-pyran was added at 10° C. Thisstep is exothermic so no additional charges should be made untilexotherm subsides. The temperature was increased to 27° C., stirred for15 min and then charged with 400 g (4.7 mol) of 3,4-dihydro-2H-pyran at24° C. Again the temperature increased (24° C. to 38° C.). The mixturewas stirred for 15 min. Once the exotherm subsided, the flask was againcharged with 400 g (4.7 mol) of 3,4-dihydro-2H-pyran at 35° C. Thetemperature again increased to 47° C. over a 20 min period. Once theexotherm subsided, the mixture was stirred for min. Finally theremaining 400 g (4.7 mol) of 3,4-dihydro-2H-pyran was added at 44° C.The temperature increased to 51° C. After stirring for one hour, asample was removed to check for removal of starting material. Uponreaction completion, contents were cooled to 20±5° C.

Example 24

Alternative Preparation of 5 from 4

To a 22.0 L 3-neck flask equipped with a stir motor, N₂ inlet, additionfunnel, cooling bath, and condenser was charged 436 g (17.96 mol) ofmagnesium turnings. 5334 g of tetrahydrofuran was then added followed by291 g (0.51 mol) of diisobutylaluminum hydride (DIBAL) (25% wt) intoluene. The mixture was stirred for 60 min at 20±5° C. Some gasevolution was seen. Next, 260-430 g˜3-5% (by weight if solution of 4 wasdropped to drums) of 4 in THF was added. The mixture was stirred for15-30 min at which time a slight exotherm should be seen (ΔT=10-15° C.).Once the exotherm was observed, the reaction mixture was cooled to 5±5°C. To this mixture, the remaining 8.22-8.39 kg of 4 in THF was added ata rate such that the temperature was kept below 30° C. (t=3 h). Thereaction was stirred at 20-25° C. for 30 min, at which time an aliquotwas removed, quench with 3 N HCl (10 mL), and analyzed.

Upon completion, the contents were cooled to −25±5° C. A solution oftrimethylborate in THF was prepared by mixing 2665 g (25.7 mol) oftrimethyl borate and 6666 g of tetrahydrofuran. This solution can beprepared in a drum with stirring.

Next, the 9331 g of trimethyl borate in THF was added at a rate suchthat the temperature was kept between −35 and −20° C. (t=2.5 h). Themixture became very thick so THF was added. After stirring at −25±5° C.for 10 min, 50 mL aliquot was removed, quenched with 25 mL of 3N HCl,and submitted for CoR. Stirring continued at −25±5° C. for 1 h, and thenthe mixture was allowed to warm to ambient temperature, where it wasstirred for at least 12 h. Pull two samples (one at 6 h and the other at12 h).

Results:

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.45-1.75 (m, 6H), 3.53 (s, 6H), 3.45(m, 1H), 3.75 (m, 1H), 4.69 (t, J=3 Hz, 1H), 4.97 (d, J=14.1 Hz, 1H),5.14 (d, J=14.1 Hz, 1H), 7.03 ((td, J=8.4, 2.7 Hz, 1H), 7.24 (dd,J=10.8, 2.1 Hz, 1H), 7.89 (t, J=7.8 Hz, 1H), 8.76 (s, 1H).

Example 25

Alternative Preparation of I from 5

To the reaction mixture above was added 5.3 kg of USP water. Afterstirring for 30 min, the mixture was charged 5.3 kg of acetic acid. Gasevolution was seen. After stirring for 30 min, an aliquot was removedfor analysis. Mixture was then heated to reflux for 36-48 hours. Duringthe reflux period, 12-13 L of THF were removed.

When the reaction was complete, the contents were cooled by the reactorto ≦40° C. by setting jacket and by charging 10.5 kg of USP water. THFwas removed until distillate did not remain. Contents of the reactorwere transferred to Rosenmund filter dryer and allowed to cool to 20±5°C. Reactor was rinsed with water, filtered, and then washed again with10.5 kg of USP water. The flask was charged with 10.5 kg of 10% ACN inwater (v/v) and agitated for 1 h. After filtering, the cake was washedwith 10.5 kg of 10% ACN in water (v/v), and then charged with 10.5 kg10% ACN in water (v/v). The contents were agitated for 1 h. The contentswere subsequently washed with 10.5 kg of USP water, charged with 7.0 Lof 5% Methyl t-Butyl Ether (MTBE)/Heptane (v/v), agitated for 1 h,filtered, charged with 7.0 L of 5% MTBE/Heptanes (v/v) and againagitated for 1 h. After filtering, the contents were charged again with7.0 L of heptane and filtered. Solids were dried at <45° C. to constantweight. Solids were recrystallized from toluene:heptane 75:25.

Example 26 Alternative Preparation of C10-Intermediate

[[4-Fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic acid

2-Bromo-5-fluorobenzyl alcohol (5 g, 24.4 mmol) was dissolved indichloromethane (100 mL). To this solution was added3,4-dihydro-2H-pyran (3.2 mL, 36.6 mmol) and (1S)-(+)-10-camphorsulfonicacid (117 mg, 0.5 mmol) and stirred at RT under nitrogen for 4 h.Saturated sodium bicarbonate was added to quench the reaction. It wasextracted using dichloromethane and the organic layer was washed withbrine and dried over sodium sulfate, then concentrated in vacuo to give[1-bromo-4-fluoro-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]benzene as acolorless oil (7 g, 100%).

[1-Bromo-4-fluoro-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]benzene (1.8g, 6.2 mmol) was dissolved in THF and cooled to −78° C. under nitrogen.To this solution was added n-butyllithium (1.6M in hexane)(6.2 mL, 9.3mmol) dropwise, then added triisopropyl borate (2.2 mL, 9.3 mmol). Themixture was slowly warmed to RT and stirred for 3 h. Water was added toquench the reaction. It was then extracted using ethyl acetate, washedwith brine, dried over sodium sulfate and concentrated in vacuo. Aftercolumn chromatography (silica gel; hexane:ethyl acetate=4:1 to 2:1)purification,[[4-Fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic acidwas obtained as a white solid (1.1 g, 70%).

Results:

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.45-1.74 (m, 6H), 3.44 (m, 1H), 3.75(m, 1H), 4.58 (d, J=13.2 Hz, 1H), 4.64 (t, J=3 Hz, 1H), 4.79 (d, J=13.2Hz, 1H), 7.03 (td, J=8.4, 2.7 Hz, 1H), 7.13 (dd, J=10.8, 2.7 Hz, 1H),7.50 (t, J=6.9 Hz, 1H).

Example 27 Alternative Preparation of C10-Intermediate

[[4-Fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic aciddimethyl ester

[[4-Fluoro-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic acid(100 mg) was dissolved in dry methanol, the solution was distilledrepeatedly to remove water. The resulting residue was immediatelycharacterized by NMR and was found to be a mixture containing dimethylester and monomethyl ester.

[[4-Fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic aciddimethyl ester

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.45-1.75 (m, 6H), 3.43 (s, 6H), 3.45(m, 1H), 3.75 (m, 1H), 4.69 (t, J=3 Hz, 1H), 4.97 (d, J=14.1 Hz, 1H),5.14 (d, J=14.1 Hz, 1H), 7.03 ((td, J=8.4, 2.7 Hz, 1H), 7.24 (dd,J=10.8, 2.1 Hz, 1H), 7.89 (t, J=7.8 Hz, 1H).

[[4-Fluoro-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronic acidmonomethyl ester

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.45-1.75 (m, 6H), 3.53 (s, 6H), 3.45(m, 1H), 3.75 (m, 1H), 4.69 (t, J=3 Hz, 1H), 4.97 (d, J=14.1 Hz, 1H),5.14 (d, J=14.1 Hz, 1H), 7.03 ((td, J=8.4, 2.7 Hz, 1H), 7.24 (dd,J=10.8, 2.1 Hz, 1H), 7.89 (t, J=7.8 Hz, 1H), 8.76 (s, 1H).

Example 28 Alternative Preparation of C10-Intermediate

[(4-Fluoro-2-methoxymethoxymethyl)phenyl]boronic acid

[1-Bromo-4-fluoro-6-methoxymethoxymethyl]benzene (525 mg, 2 mmol) wasdissolved in THF and cooled to −78° C. under nitrogen. To this solutionwas added n-butyllithium (1.6M in hexane)(1.5 mL, 2.4 mmol) dropwise,then added triisopropyl borate (0.7 mL, 2.4 mmol). The mixture wasslowly warmed to RT and stirred for 3 h. Water was added to quench thereaction. It was then extracted using ethyl acetate, washed with brine,dried over sodium sulfate and concentrated in vacuo. Afterrecrystallization from hexane:ethyl acetate=4:1[(4-Fluoro-2-methoxymethoxymethyl)phenyl]boronic acid was obtained as awhite solid (340 mg, 75%).

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 3.28 (s, 3H), 4.70 (s, 2H), 5.02 (s,2H), 7.04 (td, J=9.0, 3.0 Hz, 1H), 7.23 (dd, J=11.1, 2.4 Hz, 1H), 7.90(t, J=7.8 Hz, 1H).

Example 29 Alternative Preparation of C17-Intermediate

[[4-[4-cyanophenoxy]-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid

2-Bromo-5-(4-cyanophenoxy)benzyl alcohol (10.4 g, 34.2 mmol) wasdissolved in dichloromethane (110 mL). To this solution was added3,4-dihydro-2H-pyran (9.2 mL, 101 mmol) and (1S)-(+)-10-camphorsulfonicacid (156 mg, 0.67 mmol) and stirred at RT under nitrogen for 3 h.Methanesulfonic acid (50 μL, 0.77 mmol) was then added and reaction wasstirred overnight. Saturated sodium bicarbonate was added to quench thereaction. It was extracted using ethyl acetate and the organic layer waswashed with brine and dried over sodium sulfate, then concentrated invacuo to give[1-bromo-4-(4-cyanophenoxy)-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]benzeneas a colorless oil (13.3 g quant.).

[1-Bromo-4-(4-cyanophenoxy)-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]benzene(13.3 g, 34.2 mmol) was dissolved in THF (100 mL), triisopropyl borate(8.5 mL, 37 mmol) was added and the reaction was cooled to −78° C. undernitrogen. To this solution was added n-butyllithium (1.6M in hexane) (22mL, 35.2 mmol) dropwise. The mixture was slowly warmed to RT and stirredovernight. THF was removed in vacuo and the residue was dissolved inethyl acetate. It was then washed with water, brine, dried over sodiumsulfate and concentrated in vacuo. After column chromatography (silicagel; hexane:ethyl acetate 2:1) purification of a portion of crude,[[4-(4-cyanophenoxy)-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid was obtained as a clear oil (500 mg, 4%).

¹H-NMR (300 MHz, DMSO-d₆+D₂O) δ (ppm) 1.35-1.75 (m, 6H), 3.40 (m, 1H),3.73 (m, 1H), 4.58 (d, J=13.2 Hz, 1H), 4.59 (s, 1H), 4.77 (d, J=12.7 Hz,1H), 6.99 (dd, J=8.1, 2.2 Hz, 1H), 7.05 (m, 3H), 7.54 (d, J=7.9 Hz, 1H),7.81 (d, J=8.8 Hz, 2H).

Also isolated was[[4-(4-pentanoylphenoxy)-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid as a clear oil (500 mg, 4%). ¹H-NMR (300 MHz, DMSO-d₆+D₂O) δ(ppm)), 0.85 (t, J=7.5 Hz, 3H), 1.20-1.75 (m, 10H), 2.93 (t, J=7.0 Hz,2H), 3.42 (m, 1H), 3.70 (m, 1H), 4.58 (d, J=12.8 Hz, 1H), 4.60 (s, 1H),4.78 (d, J=13.2 Hz, 1H), 6.94 (d, J=8.4 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H),7.04 (s, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.96 (d, J=8.4 Hz, 2H).

Example 30 Alternative Preparation of C17-Intermediate

[[4-[4-cyanophenoxy]-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid dimethyl ester

Using the same method as in C10 Example IIE, a mixture of mono- anddimethyl esters of[[4-(4-cyanophenoxy)-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid were synthesized.

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.35-1.80 (m, 6H), 3.40-3.50 (m, 7H),3.60-3.70 (m, 1H), 4.43 (d, J=12.7 Hz, 1H), 4.60-4.80 (m, 2H), 6.95-7.15(m, 4H), 7.38 (d, J=8.4 Hz, 1H), 7.80-7.90 (m, 2H).

[[4-[4-cyanophenoxy]-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid monomethyl ester

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 1.35-1.80 (m, 6H), 3.40-3.50 (m, 1H),3.55 (s, 3H), 3.60-3.70 (m, 1H), 4.55 (d, J=12.8 Hz, 1H), 4.60-4.80 (m,2H), 6.95-7.15 (m, 4H), 7.53 (d, J=7.9 Hz, 1H), 7.80-7.90 (m, 2H), 8.77(s, 1H).

Using the same method as above, a mixture of mono- and dimethyl estersof[[4-(4-pentanoylphenoxy)-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid were synthesized.

[[4-(4-pentanoylphenoxy)-2-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid dimethyl ester

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 0.87 (t, J=7.3 Hz, 3H), 1.25-1.80 (m,10H), 2.94 (t, J=7.3 Hz, 2H), 3.40-3.50 (m, 7H), 3.60-3.70 (m, 1H), 4.43(d, J=12.8 Hz, 1H), 4.60-4.80 (m, 2H), 6.90-7.10 (m, 4H), 7.36 (d, J=7.9Hz, 1H), 7.95-8.05 (m, 2H).

[[4-(4-pentanoylphenoxy)-6-[(tetrahydro-2H-pyran-2-yl)oxy]methyl]phenyl]boronicacid monomethyl ester

¹H-NMR (300 MHz, DMSO-d₆) δ (ppm) 0.87 (t, J=7.3 Hz, 3H), 1.25-1.80 (m,10H), 2.94 (t, J=7.3 Hz, 2H), 3.40-3.50 (m, 1H), 3.55 (s, 3H), 3.60-3.70(m, 1H), 4.55 (d, J=12.8 Hz, 1H), 4.60-4.80 (m, 2H), 6.95-7.15 (m, 4H),7.52 (d, J=7.9 Hz, 1H), 7.95-8.05 (m, 2H), 8.75 (s, 1H).

Example 31 Alternative Preparation of C10-Intermediate

To a pre-recorded NMR tube containing a solution of2-bromo-5-fluorobenzyl alcohol (16 mg, 0.078 mmol) in CDCl₃ (0.75 mL)was injected triisopropyl borate (0.036 mL, 2 eq, 0.156 mmol) and thesolution was sonicated briefly for 30 second at room temperature. ¹H NMRdetermination indicated there were 74.3 mol % of the desiredalcohol-borate intermediate, 19.3 mol % of an unknown intermediate and6.3 mol % of unreacted alcohol.

Results:

¹H NMR (CDCl₃, 300 MHz) of (2-bromo-5-fluorobenzyl)diisopropyl borate:δ=7.45 (dd, J=8.7 Hz, J=5.1 Hz, 1H), 7.20 (dd, J=9.6 Hz, J=2.7 Hz, 1H),6.84 (td, J_(t)=8.1 Hz, J_(d)=3.3 Hz, 1H), 4.84 (s, 2H), 4.44 (septet,J=6.0 Hz, 2H), 1.18 (d, J=6.0 Hz, 12H) ppm. ¹H NMR (CDCl₃, 300 MHz) ofan unknown intermediate: δ=7.47-7.42 (1H overlap with product peaks),7.16 (dd, 1H, partially overlap with product peak), 6.91-6.81 (1H,overlap with product peak), 4.94 (s, 2H), and other unknown peaks due tooverlapping. ¹H NMR (CDCl₃, 300 MHz) of 2-bromo-5-fluorobenzyl alcoholpre-recorded before mixing: δ=7.48 (dd, J=9.0 Hz, J=5.4 Hz, 1H, overlapwith product peaks after mixing with triisopropyl borate), 7.26 (dd,J=9.3 Hz, J=3.3 Hz, 1H, intensity decreased but resolved after mixing),6.88 (td, J_(t)=8.3 Hz, J_(d)=3.0 Hz, 1H, overlap with product peaksafter mixing), 4.71 (s, 2H, CH₂ intensity decreased but resolved aftermixing), 2.04 (s, 1H, OH disappeared after mixing with triisopropylborate) ppm.

Example 32 Alternative Preparation of C17-Intermediate

The procedure described in Example II I was followed for ¹H NMRcharacterization of the current alcohol-borate intermediate. ¹H NMRdetermination indicated there were 72.7 mol % of the desiredalcohol-borate intermediate[2-bromo-5-(4-cyanophenoxy)benzyl]diisopropyl borate, 20.7 mol % of anunknown intermediate and 6.5 mol % of unreacted alcohol. ¹H NMR (CDCl₃,300 MHz) of [2-bromo-5-(4-cyanophenoxy)benzyl]diisopropyl borate: δ=7.61(d, J=9.0 Hz, 2H), 7.52 (d, J=8.4 Hz, 1H), 7.15 (d, J=3.0 Hz, 1H), 7.03(d, J=8.7 Hz, 2H), 6.84 (dd, J=8.7 Hz, J=3.0 Hz, 1H), 4.85 (s, 2H), 4.35(septet, J=6.1 Hz, 2H), 1.11 (d, J=6.1 Hz, 12H) ppm.

Example 33 Alternative Preparation

The procedure described in Example II I was followed for ¹H NMRcharacterization of the current alcohol-borate intermediate. ¹H NMRdetermination indicated there were 73.5 mol % of the desiredalcohol-borate intermediate[2-bromo-4-(4-chlorophenylthio)benzyl]diisopropyl borate, 20.2 mol % ofan unknown intermediate and 6.2 mol % of unreacted alcohol. ¹H NMR(CDCl₃, 300 MHz) of [2-bromo-4-(4-chlorophenylthio)benzyl]diisopropylborate: δ=7.48 (d, J=1.8 Hz, 1H), 7.40 (d, J=8.3 Hz, 1H), 7.27 (s, 4H),7.25 (dd, J=8.3 Hz, J=1.8 Hz, 1H), 4.86 (s, 2H), 4.42 (septet, J=6.3 Hz,2H), 1.16 (d, J=6.3 Hz, 12H) ppm.

Example 34 C10-Adenosine Complex

A mixture of 1,3-dihydro-5-fluoro-1-hydroxy-2,1-benzoxaborole (C10, 0.76g, 5 mmol), adenosine (1.34 g, 5 mmol) and sodium acetate (0.41 g, 5mmol) in dry DMF (100 mL) was stirred at 100° C. for 3 h under nitrogenatmosphere. The homogeneous solution was rotary evaporated at 50° C.under high vacuum. The residue was mixed with methylene chloride,sonicated and filtered under nitrogen atmosphere to give the desiredcomplex as white solid that was pumped overnight (2.2 g, yield 100%). ¹HNMR indicated there were 5.7 mol % of unreacted adenosine, 5.5 mol % ofunreacted C10, and the reaction conversion was more than 94%. ¹H NMR(DMSO-d₆, 300 MHz): δ=8.33 (s, 1H), 8.12 (s, 1H), 7.35-7.14 (broad m,1H), 7.29 (s, 2H), 6.80 (broad m, 1H), 6.73 (d, J=9.9 Hz, 1H), 5.99(broad d, J=2.1 Hz, 1H), 5.10 (very broad s, 1H), 4.71 (dd, J=5.7 Hz,J=3.9 Hz, 1H), 4.51 (s, 2H), 4.42 (dd, J=6.3 Hz, J=3.9 Hz, 1H), 4.07(broad s, 1H), 3.64 (dd, J=12 Hz, J=3.6 Hz, 1H) and 3.52 (dd, J=12 Hz,J=5.1 Hz, 1H) ppm; M.p: started soften at 115° C. due to residuesolvents, remained as soften solid and started decomposing at 230° C.;HPLC: 91.8% at 220 nm (adenosine was 5.3%); MS: m/z=423 (M−, ESI−), 392(M-CH₂OH, ESI+).

Example 35 C17-Adenosine Complex

The procedure described above was adapted for the preparation of thetitle complex by replacing (C10) with5-(4-cyanophenoxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (C17, 1.25 g,5 mmol). White solid product (2.7 g, yield 100%) was obtained afterpumping overnight. ¹H NMR indicated there were 3.5 mol % of unreactedadenosine, 3.5 mol % of unreacted C17, and the reaction conversion wasmore than 96%. ¹H NMR (DMSO-d₆, 300 MHz): δ=8.35 (s, 1H), 8.13 (s, 1H),7.76 (d, J=8.7 Hz, 2H), 7.45-7.36 (broad m, 1H), 7.29 (s, 2H), 7.00 (d,J=8.7 Hz, 2H), 6.81 (broad m, 1H), 6.73 (s, 1H), 6.01 (broad s, 1H),5.10 (very broad s, 1H), 4.73 (dd, J=6.0 Hz, J=3.9 Hz, 1H), 4.54 (s,2H), 4.45 (dd, J=6.0 Hz, J=3.9 Hz, 1H), 4.09 (broad s, 1H), 3.65 (dd,J=12 Hz, J=3.3 Hz, 1H) and 3.54 (dd, J=12 Hz, J=4.8 Hz, 1H) ppm; M.p:started soften at 120° C. due to residue solvents, remained as softensolid and started decomposing at 230° C.; HPLC: 92.1% at 220 nm(adenosine was 3.8%).

Example 36

C28-Adenosine Complex

The procedure for the synthesis of C10-adenosine complex was adapted forthe preparation of the title complex by replacing (C10) with6-phenylthio-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (C28, 1.21 g, 5mmol). White solid product (2.8 g, yield 100%) was obtained afterpumping overnight. ¹H NMR indicated there was 5 mol % of unreacted C28,and the reaction conversion was 95%. ¹H NMR (DMSO-d₆, 300 MHz): δ=8.29(s, 1H), 8.13 (s, 1H), 7.53 (broad s, 1H), 7.29 (s, 2H), 7.32-7.04 (m,7H), 6.05-5.96 (broad m, 1H), 5.15 (very broad s, 1H), 4.73-4.70 (m,1H), 4.58 (s, 2H), 4.46 (broad s, 1H), 4.12-4.03 (broad m, 1H), 3.63(dd, J=11.7 Hz, J=3.3 Hz, 1H) and 3.52 (dd, J=11.7 Hz, J=4.8 Hz, 1H)ppm; M.p: started soften at 110° C. due to residue solvents, remained assoften solid and started decomposing at 238° C. HPLC: 91.3% at 220 nm(adenosine was 3.8%).

Example 37 C2-Adenosine Complex

The procedure for the synthesis of C10-adenosine complex was adapted forthe preparation of the title complex by replacing (C10) with1,3-dihydro-1-hydroxy-2,1-benzoxaborole (C2, 0.67 g, 5 mmol). Creamsolid product (2.18 g, yield 100%) was obtained after pumping overnight.¹H NMR indicated there was 4.5 mol % of unreacted C2, and the reactionconversion was more than 94%. ¹H NMR (DMSO-d₆, 300 MHz): δ=8.33 (s, 1H),8.13 (s, 1H), 7.42-7.20 (broad m, 1H), 7.30 (s, 2H), 7.03-6.94 (m, 3H),6.02 (d, J=3.6 Hz, 1H), 5.25 (very broad s, 1H), 4.73 (dd, J=5.7 Hz,J=4.2 Hz, 1H), 4.56 (s, 2H), 4.46 (dd, J=6.0 Hz, J=3.9 Hz, 1H), 4.10(broad q, J=3.3 Hz, 1H), 3.66 (dd, J=12 Hz, J=2.7 Hz, 1H) and 3.52 (dd,J=11.7 Hz, J=4.8 Hz, 1H) ppm; M.p: started soften at 115° C. due toresidue solvents, remained as soften solid and started decomposing at233° C. HPLC: 91.6% at 220 nm (adenosine was 5.9%).

Example 38 Synthesis of Methyl β-D-ribofuranoside

5 g of D-Ribose was dissolved in 100 mL of methanol and cooled to 0° C.0.5 mL of concentrated sulfuric acid was added and the solution wasstored at −20° C. for 48 hrs. Solution was neutralized by passagethrough a bed of sodium carbonate and evaporated under vacuum to aviscous oil. Crude material was purified on a silica column eluting with10% methanol in ethyl acetate to yield 2.1 grams of methylβ-D-ribofuranoside.

¹H NMR 300 MHz (DMSO-d₆) δ 4.97-4.99 (d, J=4.8 Hz, 1H), 4.76-4.79 (d,J=6.6 Hz, 1H), 4.57-4.62 (m, 2H), 3.76-3.80 (m, 1H), 3.72-3.74 (m, 1H),3.66-3.71 (m, 1H), 3.44-3.50 (m, 1H), 3.26-3.34 (m, 1H), 3.19 (s, 3H)

Example 39 General Procedure for Complex Formation:

300 mg of methyl β-D-ribofuranoside was dissolved in 20 ml ofdimethylformamide. To this solution was added 1 eq of boronic ester and0.5 eq of finely powdered sodium carbonate. Reaction mixture was heatedto 100° C. and stirred for 3 hours then stripped of solvent undervacuum. Residue was co-evaporated 2 times with ethyl acetate thensonicated in dichloromethane and filtered to yield an off-white solid.

C10-Methylribose Complex

¹H NMR 300 MHz (DMSO-d₆) δ 7.28 (bs, 1H), 6.68-6.77 (m, 2H), 4.71 (s,1H), 4.52-4.55 (m, 3H), 4.26-4.28 (d, J=5.1 Hz, 1H), 4.17-4.19 (d, J=5.7Hz, 1H), 3.95-4.00 (t, J=6.8 Hz, 1H), 3.31-3.36 (m, 2H), 3.19 (s, 3H).

C17-Methylribose Complex

¹H NMR 300 MHz (DMSO-d₆) δ 7.73-7.76 (d, J=6.9 Hz, 2H), 7.38-7.41 (d,J=7.8 Hz, 1H), 6.96-6.99 (d, J=6.9 Hz, 2H), 6.72-6.75 (d, J=7.5 Hz, 1H),6.68 (s, 1H), 4.70 (s, 1H), 4.49-4.51 (m, 3H), 4.23-4.25 (d, J=5.4 Hz,1H), 4.14-4.16 (d, J=5.4 Hz, 1H), 3.95-3.98 (m, 1H), 3.22-3.26 (t,J=6.0, 1H), 3.19 (s, 3H), 3.13-3.14 (d, J=2.1, 1H).

C2-Methylribose Complex

¹H NMR 300 MHz (DMSO-d₆) δ 7.31 (bs, 1H), 6.87-6.95 (m, 3H), 4.70 (s,1H), 4.46-4.50 (m, 3H), 4.20-4.22 (d, J=5.7, 1H), 4.12-4.14 (d, J=6.0Hz, 1H), 3.94-3.99 (t, J=7.8 Hz, 1H), 3.30-3.34 (m, 2H), 3.19 (s, 3H)

C28-Methylribose Complex

¹H NMR 300 MHz (DMSO-d₆) δ 7.48 (bs, 1H), 7.21-7.26 (m, 2H), 7.05-7.12(m, 4H), 6.98-7.01 (d, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.47-4.58 (m, 3H),4.22-4.24 (d, J=5.7 Hz, 1H), 4.13-4.15 (d, J=6.0 Hz, 1H), 3.89-3.93 (t,J=6.6 Hz, 1H), 3.28-3.32 (t, J=6.5, 1H), 3.13-3.16 (m, 4H).

Example 40 Mechanism of Action

The purpose of this study is to determine the mechanism of action (MOA)of C10 in the model fungi Saccharomyces cerevisiae.

40.1 Methods

The haploid Saccharomyces cerevisiae strain ATCC 201388 was used in theselection of C10 resistant mutants. Spontaneous and EMS-inducedresistant mutants were isolated from YPD agar plates containing 4×, 8×,16×MIC of C10. All minimal inhibitory concentrations (MIC) weredetermined using NCCLS protocol M27 with the exception of using YPD orsynthetic defined media. All yeast and molecular genetic manipulationswere essentially performed as described by Guthrie C., et al., Methodsin Enzymology, 350: Part B, (2002).

40.2 Results and Conclusions

A total of 11 C10 resistant mutants were isolated from S. cerevisiae,all mutants were dominant and showed an 8 to 64-fold increase in the MICto C10. Further characterization of these mutants showed that they werenot resistant to several known antifungals including amphotericin B,cerulenin, itraconazole, aculeacin A, terbinafine, tunicamycin,ciclopirox, cyclohexamide and nikkomycin Z. All 11 mutations in the C10resistant mutants were mapped to 9 amino acid residues in the editingdomain of CDC60, the essential cytoplasmic leucyl-tRNA synthetase, oneof 40 aminoacyl-tRNA synthetases in S. cerevisiae. Furthermore, S.cerevisiae strains bearing multiple copies of CDC60 on a 2 μM plasmidwere eight times more resistant to C10. The combination of mutant andover-expression data predicts that CDC60 is the target for C10. The factthat all mutations were present in the editing domain of CDC60 indicatesthat C10 inhibits CDC60 via a novel mechanism.

The lack of a genomic sequence or any genetic tools for Trichophytonspp. makes it difficult to study the mechanism of action of C10 ineither Trichophyton species, therefore, the model fungi Saccharomycescerevisiae was used.

40.3 Materials and Methods

40.3a Chemicals, Strains and Plasmids

C10 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole, was obtained fromAnacor Pharmaceuticals, Inc. (Palo Alto, Calif., USA). All S. cerevisiaestrains and plasmids were obtained from ATCC (Manassas, Va., USA). Thehaploid Saccharomyces cerevisiae strain ATCC201388 (MATa his3 Δ1 leu2 Δ0met5 Δ0 ura3 Δ0) was used for generating mutants, while ATCC 200901(MATa leu2 Δ0 lys2 Δ0 ura3 Δ0) was used to mate with C10 resistantmutants to determine genetic dominance of the mutation. The yeast-E.coli shuttle plasmid pRS315 (Sikorski R S et al., Genetics 122: 19-27,(1989)), which has CEN6, leu2, ampR genes, and is a low copy plasmid inyeast was used in the construction of the genomic library. In theover-expression experiment, the shuttle vector pRS425 (Christianson T Wet al., Gene 110(1): 119-22 (1992)), which has the leu2 and ampR genes,and is a high copy plasmid in yeast, was used.

40.3B Isolation of Spontaneous Resistant Mutants

The haploid S. cerevisiae strain ATCC201388 was grown overnight in YPDbroth (BD, NJ USA) at 30° C. and 1 mL of cells was plated out onto YPDagar plates (YPD broth+1.5% Bacto-agar, BD, NJ USA) containing either1.6, 3.2 or 6.4 μg/mL C10 (equivalent to 4×, 8×, 16×MIC of C10).Resistant mutants appeared after 2 d incubation at 30° C. Frequency ofresistance was determined by dividing the number of resistant mutants bythe total number of cells plated as determined by plating dilutions ofthe overnight culture on YPD plates.

40.3c EMS (Ethylmethane Sulfonate) Mutagenesis

A 2.5 mL of the overnight culture, which was grown in YPD media, wascentrifuged at 700×g for 5 minutes. The cell pellet was resuspended in10 mL 50 mM potassium phosphate buffer, pH 7.0. The cell suspension wascentrifuged again and the cell pellet was resuspended in phosphatebuffer to obtain a cell density of 5×10⁷ cells/mL as determined bycounting the cells using a Petroff Hausser Counting Chamber (Horsham,Pa. USA). The cell suspension was shaken with 300 μL of EMS (Alfa Aesar,Ward Hill, Mass., USA) for 30 min. at 30° C. The mutagenesis was stoppedby adding 10% (w/v) sodium thiosulfate (Sigma-Aldrich, St. Louis, Mo.,USA), and the cells were pelleted by centrifugation at 700×g for 5 min.and resuspended in 1 mL sterile H₂O. This was repeated once more beforethe cells were plated on YPD agar plates containing 1.6 μg/mL of C10.

40.3d Determination of MICs

The minimal inhibitory concentration (MIC) was essentially performedfollowing the NCCLS guidelines outlined in the M27 protocol with theexception of using YPD or synthetic defined media (SDM).

40.3e Yeast Mating Experiment

The haploid mutants derived from S. cerevisiae ATCC201388 were mixedwith S. cerevisiae ATCC 200901, and were incubated on YPD agar plates at30° C. for 4 h. The cell mixture was streaked out on synthetic definedmedia agar (BD, NJ USA) without the amino acids lysine and methionine,which are selective for diploids.

40.3f Construction of Plasmid Genomic DNA Library

Genomic DNA from mutant strains was isolated using the DNeasy tissue kitfrom Qiagen (Valencia, Calif., USA). Genomic DNA fragments of 4-10 kbwere generated by partial digestion with Mbo I from Fermantas (Hanover,Md., USA), followed by purification using the Wizard® SV gel and PCRClean-Up system (Promega, Madison Wis. USA). The purified DNA fragmentswere ligated into pRS315 digested with BamH I (Fermantas, Hanover Md.USA) using T4 DNA ligase (Fermantas, Hanover Md. USA). The ligationmixture was dialyzed against water using the VSWP 0025 filters(Millipore, Billerica, Mass., USA) before it was electroporated intoEscherichia coli E. cloni SUPREME cells (Lucigen, Middleton, Wis., USA)following the protocol of the manufacturer. Transformants were platedout on LB plates with 200 μg/ml carbinicillin and incubated overnight at37° C. The transformants were pooled and the plasmid DNA was isolatedusing Qiagen miniprep kit (Valencia, Calif., USA). The plasmid librarywas transformed into S. cerevisiae (Gietz, R D et al., Methods inEnzymology 305: 87-96 (2002)).

40.3 g Sequencing

All sequencing was performed by Sequetech Corporation (Mountain View,Calif. USA).

40.3 g(1) Mapping Mutations

To further map the mutations to specific domains in CDC60, the followingthree pairs of primers were used 5′ gcgaaaagaaacctaacgcatattc 3′ and5′ctatcgtgatccatacaagcttgac 3′, 5′ cgatagacaatccggtgaaggtgttac 3′ and 5′catcccaaggcaatctggtacctaacc 3′, and 5′ gaaaaatacttagttgagtctttatca 3′and 5′ caccatgaggcatcttgaaatattctc 3′.

40.3h Cloning and Over-Expressing Wild Type CDC60 in S. cerevisiae

A 4.0 kb BamH I-Sal I DNA fragment containing the entire CDC60 openreading frame (ORF) and 700 bp of upstream sequence was amplified usingKOD DNA polymerase, S. cerevisiae genomic DNA (Novagen, Madison, Wis.,USA), and the primers GAG GGA TCC GGT TAG TTT TAG TTC GCG AGT GAC CTGand GAG GTC GAC GAT TTC TGG TTG CTG TTT ATT GAT CTT (Operon, Alameda,Calif., USA). This DNA fragment was then cloned into the 2 M multi-copyplasmid pRS425, and transformed into S. cerevisiae ATCC201388 (Gietz, RD et al., Methods in Enzymology 305: 87-96 (2002)).

40.4 Results and Discussions

40.4a Isolation of Resistant Mutants

From 5×10⁹ cells, 600 spontaneous C10 resistant mutants were isolated,which makes the frequency of resistance 1.2×10⁻⁷ at 4×MIC. Similarfrequencies of resistance were obtained for 8× and 16×MIC. We also usedEMS to isolate C10 resistance mutants. Use of EMS increased themutagenic frequency by 4,000 fold. The MICs of 8 spontaneous mutants and3 EMS generated mutants were tested. All the mutants showed an 8 to64-fold increase in resistance to C10 (Table 1).

TABLE 1 MICs of Spontaneous and EMS induced C10 mutants S. cerevisiaeMIC (μg/mL) Haploid Strains Cerulenin C10 ATCC201388 1 0.5 (A) 0.5 4 (B)0.5 16 (C) 0.5 16 (D) 0.5 32 (E) 0.5 16 (F) 0.5 32 (G) 0.5 32 (H) 0.5 32(I) 1 32 (J) 1 32 (K) 1 32

40.4b C10 Resistant Mutations do not Confer Resistance to OtherAntifungals

To further characterize these resistant mutants, three C10 resistantmutants were tested against various antifungal agents with knownmechanisms of action. The C10 resistant mutants did not show anyresistance to these compounds (Table 2), which suggests that C10 actsvery differently from these antifungal agents.

TABLE 2 C10 mutants are not resistant to other antifungals AntifungalMIC (μg/mL) Agents ATCC201388 (C) (G) (H) C10 0.5 16 16 16 AmphotericinB 0.125 0.125 0.125 0.125 Cyclohexamide <0.06 <0.06 <0.06 <0.06Cerulenin 0.5 0.5 0.5 0.5 Itraconazole 0.125 0.125 0.125 0.125 AculeacinA 4 4 4 4 Cicloprirox 0.5 0.5 0.5 0.5 Terbinafine 4 4 4 4 Nikkomycin Z64 64 64 64 Tunicamycin 8 8 8 8

40.4c Resistance to C10 is Dominant

In order to identify the gene that gives rise to C10 resistance, it wasfirst determined whether the mutation was either dominant or recessive.The parental S. cerevisiae strain and three mutants were selected andmated with S. cerevisiae ATCC 200901. The MIC of the diploids generatedfrom the C10 mutants were found to be 64-fold greater than the diploidgenerated from the parental strain (Table 3), which suggests that themutations are dominant, and therefore, plasmid libraries wereconstructed from these three haploid C10 resistant mutants.

TABLE 3 C10 mutants are dominant Diploid MIC (μg/mL) (mutantstrain/ATCC200901) C10 ATCC201388/ATCC200901 0.5 (C)/ATCC200901 32(G)/ATCC200901 32 (H)/ATCC200901 32

40.4d The CDC60 Gene Confers Resistance to C10

Plasmid libraries from the three mutants were transformed into S.cerevisiae ATCC201388 and selected on SDM minus leucine agar with 1μg/mL of C10. Plasmid DNA was isolated from C10 resistant transformantsand electroporated into E. coli 10G cells. The plasmid DNAs from theresulting E. coli carbenicillin resistant transformants were thentransformed into S. cerevisiae ATCC201388 to confirm that the plasmidsbore the gene for C10 resistance. One plasmid from each library thatconferred C10 resistance was sequenced and analyzed using a BLASTNsearch against the S. cerevisiae genome database (http://seq.yeastgenome.org/cgi-bin/nph-biast2sgd). The CDC60 gene was the only openreading frame identified in the cloned inserts from two plasmids derivedfrom two of the plasmid libraries. Two genes were revealed, CDC60 andPET20, in the cloned insert from the remaining plasmid library. Thissuggests that these C10 resistant mutations are located in the CDC60gene, which encodes for the cytoplasmic leucyl-tRNA synthetase. CDC60(leucyl tRNA synthetase) is one of 20 essential aminoacyl-tRNAsynthetases (ARS) that attach amino acids to the 2′ or 3′ end of tRNAs.

40.4e C10 Resistance Mutations Reside in the Editing Domain of CDC60

DNA sequence analysis of the plasmids derived from the three mutantsshowed that there was a single amino acid substitution in CDC60 fromeach of the three mutants (Table 4). An additional eight mutants wereanalyzed by amplifying CDC60 by colony PCR and transforming theresulting product into S. cerevisiae ATCC201388. All transformants wereresistant to C10 and subsequent sequence analysis showed that all ofthem contained a single amino acid change within the editing domain ofCDC60 (Table 4). The function of the ARS is to charge the correct tRNAwith the correct amino acid. In leucyl-tRNA synthetases the active sitefor the editing mechanism is located in a separate domain, which iscalled the connective polypeptide 1 (CP1), from the synthetic activesite (Schmidt E. et al, Biochemistry 34(35): 11204-10 (1995)). All ofthe amino acid substitutions from 11 mutants were located in this CP 1domain, demonstrating a link between the editing function of the enzymeand inhibitory activity of C10.

40.4f Over-Expression of Wild Type CDC60 in S. cerevisiae

Since all 11 C10 resistant mutants have single amino acid substitutionsin the editing domain of leucyl-tRNA synthetase (Table 4), it stronglysuggests that CDC60 is the target for C10. If leucyl-tRNA synthetase isthe target, increasing the copies of CDC60 should increase resistance toC10. To test this hypothesis, the wild type CDC60 gene was cloned onto amulti-copy plasmid pRS425, and transformed into S. cerevisiaeATCC201388. As shown in Table 5, the MIC for this strain is eight timeshigher than the same strain bearing pRS425.

TABLE 4 Amino acid (AA) substitutions in C10 resistant mutants Resistantmutants AA substitution in CDC60 (H) T314M (G) L315V (K) T319I (C) C326F(E) C326R (D) G405V (A) N415D (I) S416L (J) D487N (F) D487G (B) R316I

TABLE 5 CDC60 overexpression increases C10 resistance MIC (μg/mL)Compound pRS425 Plasmid control CDC60 on pRS425 (20 copies) Fluconazole2 2 1 0.125 1

Example 41

Experiments to isolate mutant leucyl tRNA transferase molecules thatwere also resistant to C10.

The haploid wild type Saccharomyces cerevisiae strain ATCC 201388 (MATahis3Δ 1 leu2Δ 0 met5Δ 0 ura3Δ 0) was used for selection of clonesshowing resistance to C10.

Mutations in the leucyl tRNA transferase were isolated in two ways. Inone set of experiments, EMS was used as a chemical mutagenic agent. 2.5mL of an overnight culture was washed 2× with 50 mM potassium phosphatebuffer, pH 7.0, and resuspended in 10 mL of the buffer to reachapproximately 5×10⁷ cells/ml. 300 μL EMS (Alfa Aesar, Ward Hill, Mass.)was added to the cells, which were then incubated for 30 min at 30° C.with shaking. The mutagenesis process was halted with the addition of10% (w/v) sodium thiosulfate (Sigma-Aldrich, St. Louis, Mo., USA). Atthe end of the mutagenesis cycle, the cells were washed 2× with waterand then plated out on YPD agar plates containing C10.

In the second method, spontaneous mutant clones were isolated from YPDplates containing large concentrations of C10. Wild type haploid S.cerevisiae strain ATCC201388 (MATa his3Δ1 leu2Δ 0 met5Δ 0 ura3Δ 0) wasgrown overnight in Difco YPD broth (1% yeast extract, 2% Bacto Peptone,2% glucose) at 30° C. to reach ˜1.0×10⁸ cells/ml. Cells wereconcentrated 10× in YPD broth, and 100 μL was plated out onto each of 30YPD agar (Difco YPD broth+1.5% Bacto agar) plates containing 1.6, 3.2,6.4 μg/ml C10 (equivalent to 4×, 8×, and 16× minimal inhibitoryconcentration of C10). Resistant mutants appeared after 2 days ofincubation at 30° C. Frequency of resistance was determined by countingthe number of the mutants, and the total number of cells.

The minimal inhibitory concentration (MIC) test was performed usingNCCLS protocol. Yeast mating experiment was conducted following theprocedure in Methods in Enzymology by Guthrie, C etc.

The genomic plasmid library for each clone was constructed using theyeast-E. coli shuttle vector pRS315 and transformed into S. cerevisiaeATCC201388. Transformants were selected on synthetic defined media with0.2 ug/ml C10 minus leucine. All sequencing work was done by Sequeteq.Blast search was performed using Saccharomyces genome database. YeastTransformation was carried out using LiAc/PEG method. Over-expression ofCDC60 construct was made by using S. cerevisiae genomic DNA, and twoprimers 5′GAGGGATCCGGTTAGT TTTAGTTCGCGAGTGACC TG 3′,5′GAGGTCGACGATTTCTGGTTGCT GTTTATTGATCTT 3′.

A total of 23 C10 resistant mutants were isolated from S. cerevisiae.All mutants were dominant and had 8-64 fold increased resistance to C10over wildtype in the minimal inhibitory concentration test. Furthercharacterization of these mutants showed that they were not crossresistant to any anti-fungal agents with known mechanism of action.

Determination of Dominance/Recessiveness

In order to identify the resistant gene in mutant strain, we firstdetermined whether the mutation is dominant or recessive. The mutant wasmated with a wild type strain with opposite mating type to make mutantdiploid. There were two sets of genes in the resulting mutant diploidcells, one from resistant mutant, and the other one from theC10-sensitive wild type. If the mutant diploid was resistant to C10, themuted gene was dominant. To map the mutation, we constructed a plasmidlibrary from the mutant strain, and transformed the library into theC10-sensitive wild type strain to select for the resistant phenotype. Ifthe mutant diploid was sensitive to C10, the muted gene would beidentified as recessive. A12, F4, H4 was mated with a wild type strain,respectively, as control; the parental strain was also mated with thesame strain. Minimal inhibitory concentrations of both wild type diploidand 3 mutant diploids are shown in Table 3. Compared to wild typediploid, all 3 mutant diploids were resistant to C10, indicating thatthe resistant mutation in these 3 mutants is dominant.

Genetic Mapping of Mutation

All the mutations in the 23 isolated C10 resistant mutants were mappedto 11 residues in the editing domain of CDC60, the cytoplasmicleucyl-tRNA synthetase.

To identify the mutation in the resistant mutant, we constructed 3plasmid genomic libraries from mutant A12, F4 and H4, respectively.Plasmids with random genomic DNA fragment insert, size from 4-10 kb,were transformed back into parental wild type strain. Transformants withplasmids carrying resistant genes were selected on SDM-leu agar plateswith addition of C10. Plasmids were then isolated and sent forsequencing. Nucleotide sequence of the insert was BLAST searched againstS. cerevisiae genome database, and the results revealed that there was asingle ORF present in the insert of both plasmids isolated from F4 andH4 plasmid library. This ORF was identified as CDC60, the cytoplasmicleucyl tRNA synthetase, one of the 20 essential cytoplasmicaminoacyl-tRNA synthetases in S. cerevisiae (there are 20 more inmitochondrial). In addition to CDC60, there was a second ORF pet20present in the plasmid isolated from A12 plasmid library, which encodedthe protein required for respiratory growth and stability of themitochondrial genome. To confirm that the CDC60 from these 3 mutantsconferred resistance to C10, we re-transformed the 3 plasmids back toparental wild type strain. Compared to the control transformation of theplasmid without CDC60, ones with CDC60 from A12, F4, H4 gave >1,000 moreresistant colonies on YPD agar containing C10, confirming that CDC60from the 3 mutant strains contributed to C10 resistance.

Sequence in CDC60 from Each of the Mutants Contains Single Amino AcidSubstitution

In order to identify whether there were any amino acid substitutions,the whole ORF of CDC60 from resistant plasmids A12, F4, and H4 wassequenced. Comparing the sequence with wild type CDC60 showed that therewas a single amino acid substitution in each of the 3 CDC60 (Table 4).In addition, sequence analysis of CDC60 from the rest of 20 resistantmutants showed that each contains a single amino acid change withinCDC60. DNA PCR fragments containing each mutation were transform backinto wild type strain. These transformations conferred resistance,indicating that the resistance of all the mutants was due to the singleamino acid substitution in CDC60.

CDC60 (leucyl tRNA synthetase) is one of the aminoacyl-tRNA synthetases(ARS) that belong to a family of essential enzymes that attach aminoacids to the 2′, or 3′ end of tRNAs, the charged tRNAs are then used inprotein synthesis. The aminoacylation of tRNA is a two-step reaction: a)activation of amino acids with ATP by forming aminoacyl adenylates andb) transferring of the aminoacyl residue from the aminoacyl adenylate tothe cognate tRNA substrate. The accuracy of aminoacylation depends onboth the specific recognition of amino acids during their activations(coarse sieve) and the pre- or post transferring editing (fine sieve).Some of the ARS have evolved editing mechanism that specificallyhydrolyzes structurally close related misactivated amino acids. LeucyltRNA synthetase is one of such enzymes that can discriminate leucinefrom isoleucine, and valine. The region that carries out this editingfunction is called connective polypeptide 1(CP1), it's a large insertionthat interrupts the active site between the third and fourth b strandsof the Rossman fold. All of the 11 amino acid substitutions from 23mutants were located in this CP1 region, suggesting that there might bea link between the editing function of the enzyme and inhibitionactivity of C10.

Example 42

Assay for Determining that C10 Inhibits the Editing Domain of tRNASynthetase in a Bacteria

This example sets forth a representative assay for determining whether aparticular compound inhibits the editing domain of an ARS in abacterium.

The [³H]-isoleucine mischarged tRNAleu was synthesized by incubating 1μM of Saccharomyces cerevisiae editing defective Cdc60p (C326F) in 500μL of 50 mM Tris-HCl (pH 8.0), 60 mM MgCl₂, 4 mM ATP, 1 mM DTT, 0.02%(w/v) BSA, 4 mg/mL crude E. coli tRNA tRNA (Roche), 0.1 mM isoleucineand 5 mCi L-[4,5-3H]isoleucine (100 Ci/mmole, GE Healthcare) and 20%(v/v) DMSO for 1 hour at 30° C. The reaction was stopped by adding 10 μLof 10% (v/v) acetic acid followed by two acidic phenol (Sigma)extractions. The mischarged tRNA in the top aqueous phase was removedand precipitated by adding two volumes of 96% (v/v) ethanol andincubating at −20° C. for 30 minutes. The precipitate was pelleted bycentrifugation at 13,200×g for 30 minutes and the mischarged tRNA pelletwas washed twice with 70% (v/v) ethanol and then resuspended in 50 mMpotassium phosphate buffer pH 5.2.

The reaction was terminated after 2 hours incubation at 30° C. by theaddition of acetic acid to 0.17% (v/v). The isoleucylated crudetRNA^(Leu) was purified by extracting twice with acidicphenol-chloroform extractions (pH 4.3), followed by ethanolprecipitation. The tRNA pellet was washed twice with 70% ethanol, driedand then resuspended in 50 mM potassium phosphate (pH 5.0) and stored at−20° C. An aliquot was precipitated with 10% (w/v) TCA to quantifyile-tRNA^(Leu).

Post-transfer editing hydrolysis assays were carried out at 30° C. in 50mM Hepes (pH 8), 10 mM MgCl₂, 30 mM KCl, with ³H-isoleucine-tRNA crude(˜0.3 μCi/mL). Each reaction was initiated by addition of the 150 nMenzyme. At each time point three 20 μL aliquots of the reaction mixturewas added to 200 μL of 10% (w/v) TCA in a Millipore filter plate andprecipitated for 20 minutes at 4° C. The precipitate was filtered andwashed three times with 200 μL of 5% (w/v) TCA, then dried and 20 μLSupermix scintillation cocktail was added. The Millipore filter plateswere counted in the MicroBeta Trilux. The IC₅₀ was determined by theamount of inhibitor that inhibited 50% activity, 100% post-transferediting was calculated by taking the activity of the no enzyme controlfrom the wild-type enzyme activity.

Compare the minimal inhibitory concentration (MIC) of a tolC Escherichiacoli strain bearing a pUC derived plasmid with and without an leuS geneinsert.

If the MIC of the strain bearing the extra copies of leuS is greaterthan 2-fold more than the control strain then pour LB agar plates withfour times the concentration of the MIC of the compound.

Plate 1×10¹⁰ E. coli on ten plates containing 4×MIC of the compound.Incubate for 1-2 days at 37° C. and pick ten colonies and restreak on4×MIC LB agar plates to confirm resistance.

Take one large colony from each of the ten E. coli resistant mutants andresuspend in 50 μL of PCR buffer.

Amplify the editing domain of CDC60 using a proof-reading PCR enzyme andthe following primers, ggcaccgtggacgtacgacaacatcgc andgggaaacaccccagtcgcgcaggcgg.

Purify the 980 bp PCR product using either Qiagen or Promega PCR cleanupkits.

Sequence amplify the mutant DNA and compared it to wild-type. If themutant DNA bears mutations in the editing domain the inhibitor affectsleucyl-tRNA synthetase via the editing domain.

Example 43

Assay for Determining that C10 Inhibits the Editing Domain of tRNASynthetase in a fun i

This example details an exemplary assay for determining whether aselected compound inhibits the editing domain of an ARS in a fungus.

The [³H]-isoleucine mischarged tRNAleu was synthesized by incubating 1μM of Saccharomyces cerevisiae editing defective Cdc60p (C326F) in 500μL of 50 mM Tris-HCl (pH 8.0), 60 mM MgCl₂, 4 mM ATP, 1 mM DTT, 0.02%(w/v) BSA, 16 M brewer's yeast tRNA (Roche), 0.1 mM isoleucine and 5 mCiL-[4,5-3H]isoleucine (100 Ci/mmole, GE Healthcare) and 20% (v/v) DMSOfor 1 hour at 30° C. The reaction was stopped by adding 10 μL of 10%(v/v) acetic acid followed by two acidic phenol (Sigma) extractions. Themischarged tRNA in the top aqueous phase was removed and precipitated byadding two volumes of 96% (v/v) ethanol and incubating at −20° C. for 30minutes. The precipitate was pelleted by centrifugation at 13,200×g for30 minutes and the mischarged tRNA pellet was washed twice with 70%(v/v) ethanol and then resuspended in 50 mM potassium phosphate bufferpH 5.2.

The reaction was terminated after 2 hours incubation at 30° C. by theaddition of acetic acid to 0.17% (v/v). The isoleucylated crudetRNA^(Leu) was purified by extracting twice with acidicphenol-chloroform extractions (pH 4.3), followed by ethanolprecipitation. The tRNA pellet was washed twice with 70% ethanol, driedand then resuspended in 50 mM potassium phosphate (pH 5.0) and stored at−20° C. An aliquot was precipitated with 10% (w/v) TCA to quantifyile-tRNA^(Leu).

Post-transfer editing hydrolysis assays were carried out at 25° C. in 50mM Hepes (pH 7.5), 10 mM MgCl₂, 30 mM KCl, with ³H-isoleucine-tRNA crude(˜0.3 μCi/mL). Each reaction was initiated by addition of the 150 nMenzyme. At each time point three 20 μL aliquots of the reaction mixturewas added to 200 μL of 10% (w/v) TCA in a Millipore filter plate andprecipitated for 20 min. at 4° C. The precipitate was filtered andwashed three times with 200 μL of 5% (w/v) TCA, then dried and 20 μLSupermix scintillation cocktail was added. The Millipore filter plateswere counted in the MicroBeta Trilux. The IC₅₀ was determined by theamount of inhibitor that inhibited 50% activity, 100% activity wascalculated by taking the activity of the no enzyme control from thewild-type enzyme post-transfer editing activity.

Example 44 Equilibrium Dialysis

Equilibrium dialysis experiments were performed in 1×AARS buffercontaining 50 mM Hepes-KOH (pH 8.0), 30 mM MgCl₂ and 30 mM KCl.Experiments were performed using 5 k MWCO DispoEquilibrium Dialyzerapparatus (Harvard Apparatus, Holliston, Mass.). On one side of thedialysis membrane (side A), [methylene-¹⁴C]C10, 2.04 GBq/mmol (Amersham)was added at concentrations ranging from 1 to 200 μM in 20 μL. On theopposite side of the membrane (side B), 30 μM recombinant Cdc60p(Saccharomyces cerevisiae cytoplasmic LeuRS) and 10 mM AMP (adenosine5′-monophosphate, Sigma) was added in 20 μL. Samples were incubated atroom temperature (21° C.) while shaking for 4.5 hrs to establish C10equilibrium across the membrane. At equilibrium, C10 on each side of thedialysis membrane was quantified by scintillation counting using aWallac MicroBeta Trilux model 1450 liquid scintillation counter. Theamount of C10 bound to Cdc60p was determined by subtracting [C10]_(A)from [C10]_(B).

PPi Exchange Assay

The PPi exchange assay was performed in 1×AARS buffer containing 50 mMHepes-KOH (pH 8.0), 30 mM MgCl₂ and 30 mM KCl supplemented with 2 mM ATPand [³²P] PPi (10⁵ cpm/μmol), 2 mM leucine and 7 nM recombinant Cdc60p.Experiments were also performed in the presence or absence of C10 (15μM) and tRNA (16 μM). After a 20 minute incubation at 30° C., reactionswere initiated by the addition of ATP. At various time intervals, 45 μLof reaction mixture was added to 100 μL of 2% perchloric acid and 0.1 MNa₄P₂O₇ to quench the reaction. Radioactive ATP was then absorbed toactivated charcoal by the addition of 30 μL of a 5% suspension ofacid-washed Norit A. This mixture was filtered though GF/C glass filtersand washed 2× with 200 μL of distilled water then 1× with 200 μL of 95%ethanol. Filters were dried and scintillation counted using a WallacMicroBeta Trilux model 1450 liquid scintillation counter.

Synthesis of Tritiated Mischarged tRNA_(leu)

The [³H]-isoleucine mischarged tRNAleu was synthesized by incubating 1μM of Saccharomyces cerevisiae editing defective Cdc60p (C326F) in 500μL of 50 mM Tris-HCl (pH 8.0), 60 mM MgCl₂, 4 mM ATP, 1 mM DTT, 0.02%(w/v) BSA, 16 μM brewer's yeast tRNA (Roche), 0.1 mM isoleucine and 5mCi L-[4,5-3H]isoleucine (100 Ci/mmole, GE Healthcare) and 20% (v/v)DMSO for 1 hour at 30° C. The reaction was stopped by adding 10 μL of10% (v/v) acetic acid followed by two acidic phenol (Sigma) extractions.The mischarged tRNA in the top aqueous phase was removed andprecipitated by adding two volumes of 96% (v/v) ethanol and incubatingat −20° C. for 30 minutes. The precipitate was pelleted bycentrifugation at 13,200×g for 30 minutes and the mischarged tRNA pelletwas washed twice with 70% (v/v) ethanol and then resuspended in 50 mMpotassium phosphate buffer pH 5.2.

Post-Transfer Editing Assay

The [³H]-isoleucine mischarged tRNAleu substrate, 40 nM, was added to 50mM Hepes-KOH pH 8.0, 30 mM KCl, 30 mM MgCl₂, 0.02% (w/v) BSA, 1 mM DTT,2.4 nM S. cerevisiae Cdc60p at 30° C. to start the reaction and 20 μLaliquots, taken at set time points, were added to ice cold 200 μL 10%(w/v) trichloroacetic acid (TCA). The TCA precipitates were washed twicewith 200 μl ice cold 5% (w/v) TCA and filtered through a Multiscreen HTSHA filter (Millipore). Optiphase (Perkin Elmer) scintillation cocktailwas added to the filters and the TCA precipitate was counted in a WallacMicroBeta Trilux model 1450 liquid scintillation counter.

Example 45

Assay for Determining that Compounds Inhibit ARS Synthesis Activity.

Aminoacylation assays were performed to determine the rate of netleucine/tRNA^(Leu) synthesis by leucyl tRNA synthetase. Experiments wereperformed in 500 ul reaction mixtures containing 1×AARS buffer (50 mMHepes-KOH (pH 8.0), mM MgCl₂ and 30 mM KCl) supplemented with 20 uM[14C]-leucine (Perkin-Elmer, 11.32 GBq/mmol.), 16 uM crude yeast tRNA,0.02% BSA, 1 mM dithiothreitol, 2 nM recombinant yeast LeuRS (CDC60) and2 mM ATP. Reactions were performed at 30 deg Celsius. At time zeroreactions were started by the addition of ATP. At various timeintervals, 20 ul aliquots were added to 150 ul of 10% trichloroaceticacid (TCA) within a single well of a 96-well nitrocelluse membranefilterplate (Millipore Multiscreen HTS, MSHAN4B50). Each well was thenwashed 3× with 100 ul of 5% TCA. Filterplates were then dried under aheat lamp and the precipitated [14C]-leucine/tRNA^(Leu) complexes werequantified by liquid scintillation counting using a Wallac MicroBetaTrilux model 1450 liquid scintillation counter. The inhibitory effectsof boron-containing compounds, was determined by addition of up to a 100uM of the compound in the reaction mixture for 20 minutes prior to theaddition of ATP.

Example 46 Test Article and Dosage Formulation

C10 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole),5-fluoro-1,3-dihydro-1-phenyl-2,1-benzoxaborole, C1(5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole), and5-fluoro-1,3-dihydro-1-(3-hydroxymethylphenyl)-2,1-benzoxaborole wereobtained from Anacor Pharmaceuticals, Inc. (Palo Alto, Calif.).[¹⁴C]-C10 was synthesized by Amersham Biosciences UK Limited(Buckinghamshire HP& 9NA, UK) radiochemical purity and specific activityof >99.3% and 55 mCi/mmol, respectively.

Penlac™ nail lacquer (ciclopirox 8% topical solution) was manufacturedby Dermik (Berwyn, Pa.). [¹⁴C]-Ciclopirox(pyridinone-6-(¹⁴C)-ciclopirox) was synthesized by PerkinElmer Life andAnalytical Sciences (Boston, Mass.). The radiochemical purity andspecific activity of the chemical was >95% and 12.5 mCi/mmol,respectively.

Experiment 1: Screening Four Oxaborole Compounds

C10, 5-fluoro-1,3-dihydro-1-phenyl-2,1-benzoxaborole, C1, and5-fluoro-1,3-dihydro-1-(3-hydroxymethylphenyl)-2,1-benzoxaborole,formulated at 10% w/v in ethanol, were tested. A single aliquot (10 μl)of each formulation was dosed to the top of human nail plates using thenail penetration procedure described below, and allowed to stand for3-days. The dosed area was washed, and then the cotton ball bedsupporting the nail and the nail samples were collected at the end ofthe incubation period, stored at 4° C. and analyzed for drug usingLC/MS/MS.

Experiment 2: Effect of Vehicle on C10 Nail Penetration

The following formulations, all containing 10% C10 were tested.Formulation A: 70% ethanol, 20% poly (vinyl methyl ether alt maleic acidmonobutyl ester (v/v); Formulation B: 6% ethanol, 14% water, 15% poly(2-hydroxyethyl methacrylate), 5% dibutyl sebacate (v/v); Formulation C:55% ethanol, 15% ethyl acetate, 15% poly (vinyl acetate), 5% dibutylsebacate (v/v); Formulation D: 20% propylene glycol, 70% ethanol (v/v).Using the nail penetration procedure described below, aliquots (10 μL)of the dose formulations were applied to human nail plates once per dayfor 14 days with a daily wash before dosing. The cotton ball bedsupporting the nail was collected from each cell chamber and replacedwith a new one at day 5, 10, and 15 after the first dose. The nailsamples were collected at the end of the 14-day dose period, stored at4° C. and analyzed for drug by LC/MS/MS.

Experiment 3: Penetration of C10 following a 14-day multiple dosetreatment

Two test articles, C10, 10% in propylene glycol and ethanol (1:4, v/v)and ciclopirox, 8% in Penlac™ nail lacquer were compared for theirpenetration rate into and through the human nail plate. Trace amounts ofcarbon-14 radiolabelled C10 and ciclopirox were added to theirrespective formulations the day before the first dose. Using the nailpenetration procedure described below, aliquots (10 μl) of the doseformulations were applied to human nail plates once per day for 14 dayswith a washing before each dose. The cotton ball bed supporting the nailwas collected from each cell chamber and replaced with a new one every72 hours after the first dose (days 3, 6, 9, 12, and 15). The nailsamples were collected at the 14-day dose period. The radioactivity ofall samples was analyzed and compared.

Nail Penetration Procedure

Details of the nail incubation have been previously described^(9, 10).Briefly, a healthy finger nail plate was mounted in a one-chamberdiffusion cell (FIG. 1, Permegear, Inc., Hellertown, Pa.) with the nailsurface (top center) open to the air and the inner surface in contactwith a small cotton ball acting as a supporting nail bed. The supportingcotton ball under the nail was wetted by normal saline providingmoisture for the nail plate, and the degree of hydration was monitoredand controlled during the experiment. The incubation period started 24hours prior to the first dose, and ended 24 hours after the final dose.Aliquots (10 μL) were applied to the surface of the nail plate oncedaily.

Dosed surface area washing was conducted at the end of incubation period(for single dose study), or each morning before dosing starting on thesecond day (multiple dose study). The dosed surface area of the nail waswashed with cotton tips in a cycle, as follows: two times with ethanol,then with 50% Ivory® liquid soap (Procter & Gamble, Cincinnati, Ohio),then two times with distilled water. The washing samples from each cyclewere pooled and the radioactivity was measured. After completion of thedosing and the incubation phase, the nail plate was transferred to acutting holder for sampling. Under the controlled humidity andtemperature, we did not observe any abnormal situations such as the nailplate color change, hydration changes, or fungal growth during the14-day dosing period. The nail plate was secured in position so that theouter dorsal-dosed surface faced the holder. The cutting holder wasmoved to bring the plate surface just barely in contact with the cuttertip. The drill was then started and a fine adjustment moved the stagetoward the cutter tip, removing a powder sample from the nail. In thisway, a hole approximately 0.3-0.4 mm in depth and 7.9 mm in diameter wasdrilled in each nail, enabling the harvest of powder sample from thecenter of each nail's ventral surface. These samples are referred to assamples taken from the “ventral/intermediate nail plate center”. Thenthe nail outside the dosing area (and also the sampling area) was cutaway and saved as the “remainder nail plate”. The layer above the powdersampling area was also saved as “the dorsal/intermediate center”. Allthe nail plate samples were individually collected into a glassscintillation vial and weighed.

Quantitative Analysis of Oxaboroles

LC/MS/MS (API3000, Applied Biosystems, Foster City, Calif.) was used toquantitate the amounts of non-radiolabeled oxaboroles, C10,5-fluoro-1,3-dihydro-1-phenyl-2,1-benzoxaborole, C1, and5-fluoro-1,3-dihydro-1-(3-hydroxymethylphenyl)-2,1-benzoxaborole insamples from the nail penetration studies. For the cotton ball analysiseleven calibration standards were prepared fresh in normal saline. Avolume of 100 μL of each standard was spiked onto a fresh cotton ballwith final calibration standard concentrations of 0, 2.5, 5, 10, 20, 40,80, 160, 320, 640, 1280, and 2560 g/mL. Acetonitrile (Burdick & Jackson,Muskegon, Mich.) containing the internal standard p-nitrophenol (PNP)was added to all cotton balls. The cotton ball samples and any residualsolvent were transferred to centrifuge filter tubes. Aftercentrifugation, the filtrate from the cotton ball samples wastransferred to autosampler vials and analyzed by LC/MS/MS. For theciclopirox samples, the filtrate was first derivatized withdimethylsulfate according to a previously described method beforeanalysis by LC/MS/MS (Myoung and Choi, 2003). Samples with calculatedconcentrations above the highest calibration standard were diluted 10-or 20-fold with acetonitrile containing internal standard p-Nitrophenol(TCI America, Portland, Oreg.). For the nail analysis, two separatecalibration curves were prepared, one for nail powder analysis and onefor top of the nail analysis. Each curve contained eleven calibrationstandards. Standards were first prepared in dimethylsulfoxide. A volumeof 10 μL of each standard was spiked onto keratin powder (6.5 mg fornail powder curve and 17 mg for top of the nail curve). Nail sampleswere digested with 1N NaOH overnight at 45° C. The next morning, beforeextraction with methylenechloride, the pH of the samples was adjusted topH 3. After extraction, the organic layer was transferred andevaporated. Samples were reconstituted in acetonitrile and analyzed byLC/MS/MS using an Eclipse XDB-C18 5 m, 2.1×50 mm column (Agilent,Wilmington, Del.) and a gradient mobile phase from 5 mM ammonium acetateand acetonitrile.

Radioactivity Measurement

All radioactivity measurements were conducted with a Model 1500 LiquidScintillation Counter (Packard Instrument Company, Downer Grove, Ill.).The counter was audited for accuracy using sealed samples of quenchedand unquenched standards as detailed by the instrument manual. The ¹⁴Ccounting efficiency is equal to or greater than 95%. All nail samplespre-treated with Packard soluene-350 were incubated at 40° C. for 48hours followed by the addition of 10 mL scintillation cocktail(HIONIC-FLUOR, Packard Instrument Company, Meriden, Conn.). Othersamples (standard dose, surface washing, and bedding material) weremixed directly with Universal ES scintillation cocktail (ICNBiomedicals, Costa Mesa, Calif.). Background control and test sampleswere counted for radioactivity for 3 minutes each.

Calculations and Data Analysis

Quantitation of non-radioactive compounds was based on peak area ratiosof compound to internal standard. The method of regression for thecalibration curves was selected based on the best fit. Linear andquadratic regression was used with 1/x or 1/x squared weighting. Allintegrations were performed using Analyst version 1.3 (AppliedBiosystems, Foster City, Calif.). The concentrations of compound in thecotton balls were converted to absolute amounts by taking the samplevolume of 100 μl into account. The amount of compound in the nail powderand top of the nail were adjusted for their respective weights andreported in g/mg.

The individual and mean (+S.E.) amount of test chemical equivalent innail, bedding material, and wash samples are presented as dpm, Ci,percent administered dose, and mg equivalent at each time point. Theconcentration of ¹⁴C-labeled test chemicals were calculated from thevalue based on the specific activity of each [¹⁴C]-labeled testchemical. The information of concentration of non-labeled test chemicalin the topical formulation was obtained from the manufacturers. Thetotal concentration of test chemical equivalent is the sum of theconcentration of ¹⁴C-labeled test chemical and the concentration ofnon-labeled test chemical. The value of the total amount of testchemical equivalent in each nail sample was calculated from those valuesbased on the radioactivity of the sample and the ratio of total mg testchemical equivalent and radioactivity of the test chemical. The data wasfurther normalized by dividing by the weight of the sample. Statisticalsignificant of nail samples from every two groups was analyzed bystudent t-test.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-192. (canceled)
 193. A pharmaceutical formulation comprising: a) apharmaceutically acceptable excipient which is alcohol; b) a compoundwhich penetrates the nail plate and travels through the nail plate to anail bed underlying said nail plate; c) an emollient which is a cyclicpolydimethylsiloxane containing from about 3 to about 9 silicon atoms;and having an efficacy coefficient against a Trichophyton species ofbetween about 10 and about 1000; wherein the compound is present in saidpharmaceutical formulation in a concentration of from about 0.5% toabout 15%; and wherein said pharmaceutical formulation is for ungualadministration to a human suffering from onychomycosis mediated at leastin part by said Trichophyton species.
 194. The formulation of claim 193,wherein the compound is present in said pharmaceutical formulation in aconcentration of from about 1% to about 10%.
 195. The formulation ofclaim 193, wherein the cyclic polydimethylsiloxane is cyclomethicone.196. The formulation of claim 193, further comprising an emollient whichis diisopropyl adipate.
 197. The formulation of claim 193, wherein theemollient is present in an amount of from about 10% to about 50%. 198.The formulation of claim 193, wherein said pharmaceutically acceptableexcipient is propanol or isopropanol.
 199. The formulation of claim 193,wherein said pharmaceutically acceptable excipient is ethanol.
 200. Theformulation of claim 193, further comprising an antioxidant.
 201. Theformulation of claim 200, wherein said antioxidant is vitamin E.